Fate of Salmonella Senftenberg in broiler chickens evaluated by challenge experiments

Abstract

Experimental and epidemiological evidence has indicated the respiratory route to be a potential portal of entry for salmonellas in poultry. The purpose of this study was to evaluate and compare the infectivity of Salmonella enterica serovar Senftenberg following oral gavage, intratracheal or intravenous challenge in chickens. Seven-day-old chicks were challenged with either 10⁴ or 10⁶ colony-forming units of S. Senftenberg per chick by oral gavage, intratracheal or intravenous challenge, respectively, in two independent trials. Chickens were humanely killed 24 h post challenge and S. Senftenberg was cultured and enumerated from caecal contents, caecal tissue–caecal tonsils and liver and spleen. In both trials, intratracheal delivery of S. Senftenberg was the only route that allowed colonization of the caeca of chickens when compared with oral gavage or intravenous challenge in a dose response fashion (P < 0.05). Liver and spleen samples yielded no S. Seftenberg after the lower dose challenge by the oral or intratracheal route and only low levels following the high-dose administration by these routes, whereas intravenous challenge resulted in recovery of the organisms after both doses. The results of the present study suggest that S. Senftenberg entering the blood is likely to be cleared and will not be able to colonize caeca to the same extent as compared with intratracheal challenge. Clarification of the potential importance of the respiratory tract for transmission of salmonellas under field conditions may be of critical importance to develop intervention strategies to reduce the transmission in poultry.

Introduction

The natural route of transmission of zoonotic pathogens such as salmonellas is oral–faecal (White et al., 1997; Galanis et al., 2006); however, studies have also suggested that airborne transmission of salmonellas in poultry is possible (Mitchell et al., 2002; Albrecht et al., 2003; Harbaugh et al., 2006; López et al., 2012). The frequent recovery of salmonellas from dust and bioaerosols from infected poultry is well documented (Mitchell et al., 2002; Lues et al., 2007; Chinivasagam et al., 2009) and bioaerosolized salmonellas of a very small droplet size are capable of reaching the lower respiratory tract of chicks (Mensah & Brain, 1982; Cox & Pavic, 2010). Members of Salmonella are viable in laboratory-generated aerosols for more than 2 to 4 h depending on the overall prevailing relative humidity, the temperature of the air, and the strain (Wathes et al., 1988; McDermid & Lever, 1996). A detailed study by Hayter & Besch (1974) showed that the site of deposition of particles critically depends on their size. Large respired particles with a size of 4 to 7 µm get trapped and removed by the mucociliary ladder, but particles smaller than this are deposited in deeper areas of the respiratory organs. Aerosols of size <4 µm should be able to carry salmonellas, considering that their diameters are typically between 0.7 and 1.5 µm and lengths from 2 to 4 µm, and hence would not face the mucociliary challenge. Several studies have analysed the relationship between the particles in the air, both size and number, and the dose of salmonellas, with estimates ranging from 10 colony-forming units (CFU) to 1.2 × 10⁴ CFU of salmonellas/m³ of air, depending on the conditions (Baskerville et al., 1992; Radon et al., 2002).

Our laboratory has recently hypothesized that transmission by the faecal–respiratory route may be a viable portal of entry for salmonellas. This mode of infection could explain some clinical impressions of relatively low-dose infectivity under field conditions in relation to the high oral challenge dose that is typically required for infection through the oral route in laboratory studies. Recently published results from our laboratory compared intratracheal (i.t.) versus oral administration of two non-typhoid serovars of Salmonella enterica, S. Enteritidis and S. Typhimurium, with relatively different invasiveness (Kallapura et al., 2014b). Neonate chicks were infected via the respiratory route at a very low dose with caecal colonization that was equivalent to the salmonella recovered from a higher oral challenge (Kallapura et al., 2014a). These results may be important considering previous studies (Berrang et al., 1995) demonstrating fan-driven spread of salmonellas within the hatching cabinet and hatchery incubators. Previous studies to date have not described the subsequent fate of salmonellas infecting through the respiratory route (Stearns et al., 1987; Wathes et al., 1988; Nganpiep & Maina, 2002; Kallapura et al., 2014a). However, the ability of salmonellas to reach the liver and spleen (LS) and to colonize caeca when administered intratracheally suggested a possible route of systemic infection (Kallapura et al., 2014b). The purpose of the present study was to evaluate the fate and dissemination of S. Senftenberg post oral, i.t. or intravenous (i.v.) challenge in broiler chickens.

Materials and Methods

Experimental birds

Day-of-hatch broiler chickens were obtained from Cobb-Vantress (Siloam Springs, AZ, USA) and were placed in isolators, in a controlled age-appropriate environment. Chicks were provided ad libitum access to water and a balanced unmedicated corn–soybean diet meeting the nutrition requirements of poultry (National Research Council, 1994). All bird-handling procedures were in compliance with the Institutional Animal Care and Use Committee at the University of Arkansas. A small number of chicks (n = 12), for each trial, were humanely killed upon arrival. The caecal tissue with caecal tonsils (CCT), LS, and trachea were aseptically removed, individually cultured in tetrathionate enrichment broth (Becton Dickinson, Sparks, MD, USA), and confirmed negative for salmonellas by plating the samples on Xylose Lysine Tergitol-4 (XLT-4; BD diagnostics, Difco, Detroit, Michigand, USA) selective medium.

Salmonella cultures

The challenge strain of S. Senftenberg was obtained from a field case. The identity was established to sero-group by agglutination with O factor 19 antiserum (Becton Dickinson) and typed by the National Veterinary Services Laboratories (Ames, IA, USA). One hundred microlitres of a frozen aliquot of S. Senftenberg were added to 10 ml tryptic soy broth (Becton Dickinson). The broth was incubated for 8 h at 37°C. This was followed by three passages every 8 h into fresh tryptic soy broth, for a total of 24 h, to ensure that the culture was in log phase. A sample of the tryptic soy broth from each passage was cultured onto XLT-4 to ensure purity. Post incubation, salmonellas were washed three times in sterile 0.9% saline by centrifugation (1864 × g, 4°C, 15 min), quantified with a spectrophotometer (Spectronic 20D+; Spectronic Instruments, Thermo Scientific, Madison, Wisconsin, USA) at 625 nm and diluted in sterile 0.9% saline as per required concentrations (CFU/ml) for the trials. Concentrations of S. Senftenberg were also determined retrospectively by serial dilution and further plating on XLT-4 medium for enumeration of actual CFU/ml used for challenge.

Challenge

Birds were challenged with high or low doses administered orally, intratracheally, or intravenously in two independent trials. S. Senftenberg oral and i.t. challenges were given using sterile gavage needles with a 22-gauge stainless steel assembly of tubing and ball (1.25 mm diameter), in a volume of 0.25 ml. Care was taken, while challenging chicks intratracheally, to insert the gavage needle accurately into the trachea as deep as possible and discharge the challenge near the bifurcation of the trachea. Chicks were challenged intravenously using sterile 25-gauge needles (Becton Dickson). The jugular vein, which is considered the best access point to the peripheral circulation, was chosen for the 0.25 ml volume i.v. injections.

Experimental design

Ninety day-of-hatch broiler chicks were obtained, randomly assigned to six groups (n = 15 chickens per group), and placed in isolators by group with unrestricted access to feed and water. On day 7, groups were challenged with either 10⁴ or 10⁶ CFU of S. Senftenberg per chick, orally, intratracheally or intravenously, respectively, in two independent trials. All chicks were humanely killed 24 h post challenge and 12 chicks/group were cultured for enumeration of S. Senftenberg from caecal contents, as well as organ enrichment (CCT and LS) on selective XLT-4 medium.

Data and statistical analysis

Data from the trials were subjected to analysis of variance (SAS, Cary, NC, USA). Log₁₀ CFU values of S. Senftenberg per gram of caeca were expressed as mean ± standard error of the mean and deemed significant if P ≤ 0.05. The data were also subjected to mean separation using Duncan's multiple-range test at a 5% level of significance. The enrichment data were expressed as positive/total chickens (%) and the percent recovery of S. Senftenberg was compared using the chi-squared test of independence, testing all possible combinations to determine the significance (P ≤ 0.05) for these studies (Zar, 1984).

Results and Discussion

Poultry are a major reservoir for food-borne Salmonella serovars with S. Typhimurium, S. Enteritidis, S. Heidelberg, S. Kentucky and S. Senftenberg being the most prevalent serovars in the USA (Centers for Disease Control and Prevention, 2008; FDA, 2010). The same serovars are also linked to leading causes of food-borne illness worldwide (Scallan et al., 2011). In chickens, infections with the host-specific serovars S. Gallinarum and S. Pullorum cause septicaemic fowl typhoid and pullorum disease, respectively (Barrow & Freitas Neto, 2011), whereas infections with serovars without host specificity generally display no clinical signs. In the last decade, significant progress has been made in the knowledge of invasion and pathogenesis of salmonellas in mammalian hosts; however, information regarding invasion and colonization mechanisms and interactions with host cells in poultry is limited and poorly defined (He et al., 2012). S. Senftenberg, a serovar that is more resistant to the environmental stresses, is frequently isolated from hatching houses and raw feed materials, and is adapted to colonize and persist in poultry houses (Pedersen et al., 2008). The prevailing theory of enteropathogenesis of salmonellas is that bacterial invasion of the intestinal epithelium is essential for virulence and that this requires the virulence-associated genomic region Salmonella pathogenicity island 1 (Ochman & Groisman, 1996; Hensel, 2004). However, S. Senftenberg strains isolated from food-borne outbreaks have been reported to lack the invasion-associated locus Salmonella pathogenicity island 1, indicating that Salmonella pathogenicity island 1 is not essential for intestinal inflammatory disease (Hu et al., 2008). Yet, despite its lack of invasiveness, S. Senftenberg has emerged as a human pathogen, with the ability to acquire important pathogenic loci from other bacteria, such as the Yersinia high pathogenicity island, which encodes a yersiniabactin-mediated iron-acquisition system present in highly pathogenic strains of Yersinia and several members of the Enterobacteriaceae (Petermann et al., 2008). Several studies have illustrated the presence of the high pathogenicity island in S. Senftenberg and the importance of poultry and pigs as a reservoir and vehicle for the dissemination of zoonotic pathogens (Ginocchio et al., 1997; Hacker et al., 1997; Carniel, 2001; Arnold et al., 2004).

The results of the evaluation of oral, i.t., or i.v. challenge of chickens with S. Senftenberg from Trials 1 and 2 are summarized in Table 1. It was remarkable to observe that i.t. delivery of S. Senftenberg was the only route able to colonize the caeca of the chickens when compared with oral or i.v. challenge in a dose-response fashion in both trials (P < 0.05). The low levels of S. Senftenberg recovery from selectively enriched LS samples observed only after a high-dose administration by the oral or intratracheal route (Table 1) are in agreement with previous reports suggesting the non-invasive properties of S. Senftenberg (Hu et al., 2008; He et al., 2012). Taken together, the results from this study may suggest that systemic infection, leading to biliary clearance (Lee et al., 1981), was responsible for the intestinal infections from the i.t.-challenged chicks, although other mechanisms are possible. These results were in alignment with our previous results (Kallapura et al., 2014b) and suggested that salmonellas, depending on the strain, were far more likely to be recovered from CCT when administered intratracheally, presumably involving a systemic dissemination and infection in chickens. Furthermore, previous studies to date have not described the subsequent fate of salmonellas infecting the respiratory system, while evaluating the infection via the respiratory route (Stearns et al., 1987; Wathes et al., 1988; Nganpiep & Maina, 2002; Kallapura et al., 2014a). The increased S. Senftenberg recovery and enumeration from caeca following i.t. administration in Trials 1 and 2 suggested that challenge by the i.t. route might be more effective for colonization of 7-day-old chicks (Table 1).

Table 1. Evaluation of oral, intratracheal or intravenous challenge of chickens with Salmonella enterica serovar Senftenberg on caecal colonization and organ invasion.

		Trial 1			Trial 2
Challenge dose (CFU/chick)	Route of challenge	Log10 S. Senftenberg/g caeca content	Caeca tissue–caecal tonsil	Liver and spleen	Log10 S. Senftenberg/g caeca content	Caeca tissue–caecal tonsil	Liver and spleen
10^4	Oral	0.0 ± 0.0^C	0/12 (0%)^B	0/12 (0%)^B	0.0 ± 0.0^C	0/12 (0%)^B	0/12 (0%)^B
10^4	Intratracheal	0.90 ± 0.38^B	4/12 (33.3%)^A	0/12 (0%)^B	1.700 ± 0.47^B	5/12 (41.6%)^A	0/12 (0%)^B
10^4	Intravenous	0.0 ± 0.0^C	0/12 (0%)^B	5/12 (41.6%)^A	0.0 ± 0.0^C	0/12 (0%)^B	6/12 (50.0%)^A
10^6	Oral	0.0 ± 0.0^C	0/12 (0%)^B	1/12 (8.3%)^B	0.0 ± 0.0^C	0/12 (0%)^B	2/12 (16.6%)^B
10^6	Intratracheal	2.47 ± 0.22^A	11/12 (91.6%)^A	2/12 (16.6%)^B	2.25 ± 0.38^A	10/12 (83.3%)^A	3/12 (25.0%)^B
10^6	Intravenous	0.0 ± 0.0^C	0/12 (0%)^B	9/12 (75.0%)^A	0.0 ± 0.0^C	0/12 (0%)^B	10/12 (83.3%)^A

Enumeration data of log₁₀ S. Senftenberg per gram of caeca content are expressed as mean ± standard error of the mean. Recovery incidence data are expressed as positive/total for each tissue sampled (%). Different uppercase superscript letters within columns indicate significant differences P < 0.05, n = 12/group.

One possible method of systemic spread and caecal colonization of S. Senftenberg when given intratracheally is that salmonellas, conceivably reaching the non-ciliated regions of the respiratory tract, might either be able to translocate directly into the blood or be directly phagocytized through several documented mechanisms for removing particulate matter from the non-ciliated avian respiratory tract (Fagerland & Arp, 1993). The mode in which salmonellas infect and spread systemically by directly entering the blood directly and causing bacteraemia is quite conceivable considering previous studies proposing disrupted epithelial barrier of the developing bronchus-associated lymphoid tissue structure, endocytosis by epithelial cells and further transport to the interstitial matrix and the high prevalence of blood–gas barrier breaks and epithelial–epithelial cell connection breaks (West & Mathieu-Costello, 1995). Alternatively, various reports have described the presence of an efficient phagocytic system in chickens, involving phagocytic epithelial lining, interstitial macrophages in the blood–gas barrier and the infiltrating free avian respiratory macrophages (Mensah & Brain, 1982). Continued survival of bacteria entering blood is considered unlikely. Reports suggest that blood-borne bacteria are rapidly cleared from the circulation and the clearance rate is enhanced significantly in the presence of opsonic factors in the blood (Qureshi et al., 2000). Passively immunized turkey poults when challenged with Escherichia coli rapidly reduced the bacterial load by approximately 5 logs within 10 min post i.v. challenge, in contrast to the non-immunized poults (Arp, 1982). In addition, a study by Kiama et al. (2008) demonstrated that chicken lung lavaged erythrocytes readily phagocytized 1.5 µm diameter polystyrene particles compared with rat erythrocytes, which showed absolutely no signs of phagocytosis. Even though little is known about such interactions, phagocytic activity by chicken erythrocytes is attributed to the nucleated properties (Kiama et al., 2008). The role(s) that the inherently phagocytic erythrocytes might play, particularly in the defence of the pulmonary system and the avian body in general, needs to be confirmed with further studies.

Salmonellas have an established intracellular lifestyle and are known to disseminate systemically via macrophages when given orally (Vazquez-Torres et al., 2000), and could potentially infect chickens via a similar pathway following respiratory exposure. Not surprisingly, S. Senftenberg was recovered in a dose-related fashion from the LS of chickens after i.v. administration in both experiments. However, S. Senftenberg was not recovered after enrichment of CCT even with a high i.v. dose in either experiment.

Overall, the results of the present study demonstrate that salmonellas entering the blood are likely to be cleared and will not be able to colonize the caeca to the same extent as i.t. challenge (Table 1), hence suggesting the need for macrophage-mediated systemic dissemination studies of the respiratory tract. The possibility that respiratory transmission is apparently more effective than the oral–faecal route of infection might explain the relative difficulty in consistently infecting chicks via oral administration under laboratory conditions, as opposed to the apparent ease of transmission under commercial conditions, and is an intriguing concept. Ongoing studies are aimed at further evaluation of this hypothesis using bioaerosol administration. Clarification of the potential importance of the respiratory tract for transmission of salmonellas under field conditions may be of critical importance to develop intervention strategies to reduce its transmission in poultry.