Assessing the potential impact of Salmonella vaccines in an endemically infected dairy herd

Salmonella spp. in cattle contribute to bacterial foodborne disease for humans. Reduction of Salmonella prevalence in herds is important to prevent human Salmonella infections. Typical control measures are culling of infectious animals, vaccination, and improved hygiene management. Vaccines have been developed for controlling Salmonella transmission in dairy herds; however, these vaccines are imperfect and a variety of vaccine effects on susceptibility, infectiousness, Salmonella shedding level, and duration of infectious period were reported. To assess the potential impact of imperfect Salmonella vaccines on prevalence over time and the eradication criterion, we developed a deterministic compartmental model with both replacement (cohort) and lifetime (continuous) vaccination strategies, and applied it to a Salmonella Cerro infection in a dairy farm. To understand the uncertainty of prevalence and identify key model parameters, global parameter uncertainty and sensitivity analyses were performed. The results show that imperfect Salmonella vaccines reduce the prevalence of Salmonella Cerro. Among three vaccine effects that were being considered, decreasing the length of the infectious period is most effective in reducing the endemic prevalence. Analyses of contour lines of prevalence or the critical reproduction ratio illustrate that, reducing prevalence to a certain level or zero can be achieved by choosing vaccines that have either a single vaccine effect at relatively high effectiveness, or two or more vaccine effects at relatively low effectiveness. Parameter sensitivity analysis suggests that effective control measures through applying Salmonella vaccines should be adjusted at different stages of infection. In addition, lifetime (continuous) vaccination is more effective than replacement (cohort) vaccination. The potential application of the developed vaccination model to other Salmonella serotypes related to foodborne diseases was also discussed. The presented study may be used as a tool for guiding the development of Salmonella vaccines.     

TLR4 single nucleotide polymorphisms (SNPs) associated with Salmonella shedding in pigs

Toll-like receptor 4 (TLR4) is a key factor in the innate immune recognition of lipopolysaccharide (LPS) from Gram-negative bacteria. Previous studies from our group identified differences in the expression profile of TLR4 and genes affected by the TLR4 signaling pathway among pigs that shed varying levels of Salmonella, a Gram-negative bacterium. Therefore, genetic variation in this gene may be involved with the host’s immune response to bacterial infections. The current study screened for single nucleotide polymorphisms (SNPs) in the TLR4 gene and tested their association with Salmonella fecal shedding. Pigs (n?=?117) were intranasally challenged at 7 weeks of age with 1?×?10⁹ CFU of S. Typhimurium χ4232 and were classified as low or persistent Salmonella shedders based on the levels of Salmonella being excreted in fecal material. Salmonella fecal shedding was determined by quantitative bacteriology on days 2, 7, 14, and 20/21 post exposure, and the cumulative levels of Salmonella were calculated to identify the low (n?=?20) and persistent (n?=?20) Salmonella shedder pigs. From those 40 animals, the TLR4 region was sequenced, and 18 single nucleotide polymorphisms (SNPs) in TLR4 were identified. Twelve SNPs have been previously described and six are novel SNPs of which five are in the 5′ untranslated region and one is in intron 2. Single marker association test identified 13 SNPs associated with the qualitative trait of Salmonella fecal shedding, and seven of those SNPs were also associated with a quantitative measurement of fecal shedding (P?<?0.05). Using a stepwise regression process, a haplotype composed of SNPs rs80787918 and rs80907449 (P?≤?4.0?×?10^?3) spanning a region of 4.9 Kb was identified, thereby providing additional information of the influence of those SNPs on Salmonella fecal shedding in pigs.     

Sensitivity analysis to identify key parameters influencing Salmonella infection dynamics in a pig batch

In the context of managed herds, epidemiological models usually take into account relatively complex interactions involving a high number of parameters. Some parameters may be uncertain and/or highly variable, especially epidemiological parameters. Their impact on the model outputs must then be assessed by a sensitivity analysis, allowing to identify key parameters. The prevalence over time is an output of particular interest in epidemiological models, so sensitivity analysis methods adapted to such dynamic output are needed.In this paper, such a sensitivity analysis method, based on a principal component analysis and on analysis of variance, is presented. It allows to compute a generalised sensitivity index for each parameter of a model representing Salmonella spread within a pig batch. The model is a stochastic discrete-time model describing the batch dynamics and movements between rearing rooms, from birth to slaughterhouse delivery. Four health states were introduced: Salmonella-free, seronegative shedder, seropositive shedder and seropositive carrier. The indirect transmission was modelled via an infection probability function depending on the quantity of Salmonella in the rearing room.Simulations were run according to a fractional factorial design enabling the estimation of main effects and two-factor interactions. For each of the 18 epidemiological parameters, four values were chosen, leading to 4096 scenarios. For each scenario, 15 replications were performed, leading to 61 440 simulations. The sensitivity analysis was then conducted on the seroprevalence output.The parameters governing the infection probability function and residual room contaminations were identified as key parameters. To control the Salmonella seroprevalence, efficient measures should therefore aim at these parameters. Moreover, the shedding rate and maternal protective factor also had a major impact. Therefore, further investigation on the protective effect of maternal or post-infection antibodies would be needed.     

Gut microbiota composition before infection determines the Salmonella super- and low-shedder phenotypes in chicken

Heterogeneity of infection and extreme shedding patterns are common features of animal infectious diseases. Individual hosts that are super-shedders are key targets for control strategies. Nevertheless, the mechanisms associated with the emergence of super-shedders remain largely unknown. During chicken salmonellosis, a high heterogeneity of infection is observed when animal-to-animal cross-contaminations and reinfections are reduced. We hypothesized that unlike super-shedders, low-shedders would be able to block the first Salmonella colonization thanks to a different gut microbiota. The present study demonstrates that (i) axenic and antibiotic-treated chicks are more prone to become super-shedders; (ii) super or low-shedder phenotypes can be acquired through microbiota transfer; (iii) specific gut microbiota taxonomic features determine whether the chicks develop a low- and super-shedder phenotype after Salmonella infection in isolator; (iv) partial protection can be conferred by inoculation of four commensal bacteria prior to Salmonella infection. This study demonstrates the key role plays by gut microbiota composition in the heterogeneity of infection and pave the way for developing predictive biomarkers and protective probiotics.     

Toll-like Receptor 11 (TLR11) Prevents Salmonella Penetration into the Murine Peyer Patches

Toll-like receptors (TLRs) are key molecular sensors used by the mammalian innate immune system to detect microorganisms. Although TLR functions in colonic immune homeostasis and tolerance to commensal bacteria have been intensively researched, the precise roles of different TLRs in response to pathogen infection in the gut remain elusive. Peyer patches are the major entrance of Salmonella infection and antigen transportation in intestine. Here, we report that, in contrast to TLR5 as a “carrier of Salmonella,” TLR11 works as a “blocker of Salmonella” to prevent highly invasive Salmonella from penetrating into the murine Peyer patches and spreading systemically. TLR11 plays an important role in mediating TNF-α induction and systemic inflammation in response to Salmonella infection. Remarkably, in mice lacking TLR11, apparent hemorrhages at Peyer patches are induced by highly invasive Salmonella, a phenotype resembling human Salmonella infection. Therefore, our results indicate a potentially important role for TLR11 in preventing murine intestinal infection and modulating antigen transportation in the gut and imply an important role for various TLRs in cooperation with tight control of pathogens penetrating into Peyer patches. The TLR11 knock-out mouse can serve as a good animal model to study Salmonella infection.     

An antibody targeting type III secretion system induces broad protection against Salmonella and Shigella infections

Salmonella and Shigella bacteria are food- and waterborne pathogens that are responsible for enteric infections in humans and are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of strains resistant to antibiotics requires the development of broadly protective therapies. Recently, the needle tip proteins of the type III secretion system of these bacteria were successfully utilized (SipD for Salmonella and IpaD for Shigella) as vaccine immunogens to provide good prophylactic cross-protection in murine models of infections. From these experiments, we have isolated a cross-protective monoclonal antibody directed against a conserved region of both proteins. Its conformational epitope determined by Deep Mutational Scanning is conserved among needle tip proteins of all pathogenic Shigella species and Salmonella serovars, and are well recognized by this antibody. Our study provides the first in vivo experimental evidence of the importance of this common region in the mechanism of virulence of Salmonella and Shigella and opens the way to the development of cross-protective therapeutic agents.     

Specific Monoclonal Antibody Overcomes the Salmonella enterica Serovar Typhimurium’s Adaptive Mechanisms of Intramacrophage Survival and Replication

Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb)-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria.     

Independent Bottlenecks Characterize Colonization of Systemic Compartments and Gut Lymphoid Tissue by Salmonella

Vaccination represents an important instrument to control typhoid fever in humans and protects mice from lethal infection with mouse pathogenic serovars of Salmonella species. Mixed infections with tagged Salmonella can be used in combination with probabilistic models to describe the dynamics of the infection process. Here we used mixed oral infections with tagged Salmonella strains to identify bottlenecks in the infection process in naïve and vaccinated mice. We established a next generation sequencing based method to characterize the composition of tagged Salmonella strains which offers a fast and reliable method to characterise the composition of genome-tagged Salmonella strains. We show that initial colonization of Salmonella was distinguished by a non-Darwinian selection of few bacteria setting up the infection independently in gut associated lymphoid tissue and systemic compartments. Colonization of Peyer''s patches fuels the sustained spread of bacteria into mesenteric lymph nodes via dendritic cells. In contrast, infection of liver and spleen originated from an independent pool of bacteria. Vaccination only moderately reduced invasion of Peyer''s patches but potently uncoupled bacterial populations present in different systemic compartments. Our data indicate that vaccination differentially skews the capacity of Salmonella to colonize systemic and gut immune compartments and provide a framework for the further dissection of infection dynamics.     

Integrating Survey and Molecular Approaches to Better Understand Wildlife Disease Ecology

Infectious wildlife diseases have enormous global impacts, leading to human pandemics, global biodiversity declines and socio-economic hardship. Understanding how infection persists and is transmitted in wildlife is critical for managing diseases, but our understanding is limited. Our study aim was to better understand how infectious disease persists in wildlife populations by integrating genetics, ecology and epidemiology approaches. Specifically, we aimed to determine whether environmental or host factors were stronger drivers of Salmonella persistence or transmission within a remote and isolated wild pig (Sus scrofa) population. We determined the Salmonella infection status of wild pigs. Salmonella isolates were genotyped and a range of data was collected on putative risk factors for Salmonella transmission. We a priori identified several plausible biological hypotheses for Salmonella prevalence (cross sectional study design) versus transmission (molecular case series study design) and fit the data to these models. There were 543 wild pig Salmonella observations, sampled at 93 unique locations. Salmonella prevalence was 41% (95% confidence interval [CI]: 37–45%). The median Salmonella DICE coefficient (or Salmonella genetic similarity) was 52% (interquartile range [IQR]: 42–62%). Using the traditional cross sectional prevalence study design, the only supported model was based on the hypothesis that abundance of available ecological resources determines Salmonella prevalence in wild pigs. In the molecular study design, spatial proximity and herd membership as well as some individual risk factors (sex, condition score and relative density) determined transmission between pigs. Traditional cross sectional surveys and molecular epidemiological approaches are complementary and together can enhance understanding of disease ecology: abundance of ecological resources critical for wildlife influences Salmonella prevalence, whereas Salmonella transmission is driven by local spatial, social, density and individual factors, rather than resources. This enhanced understanding has implications for the control of diseases in wildlife populations. Attempts to manage wildlife disease using simplistic density approaches do not acknowledge the complexity of disease ecology.     

Modelling the effect of heterogeneity of shedding on the within herd Coxiella burnetii spread and identification of key parameters by sensitivity analysis

Coxiella burnetii is the bacterium responsible for Q fever, a worldwide zoonosis. Ruminants, especially cattle, are recognized as the most important source of human infections. Although a great heterogeneity between shedder cows has been described, no previous studies have determined which features such as shedding route and duration or the quantity of bacteria shed have the strongest impact on the environmental contamination and thus on the zoonotic risk. Our objective was to identify key parameters whose variation highly influences C. burnetii spread within a dairy cattle herd, especially those related to the heterogeneity of shedding.To compare the impact of epidemiological parameters on different dynamical aspects of C. burnetii infection, we performed a sensitivity analysis on an original stochastic model describing the bacterium spread and representing the individual variability of the shedding duration, routes and intensity as well as herd demography. This sensitivity analysis consisted of a principal component analysis followed by an ANOVA. Our findings show that the most influential parameters are the probability distribution governing the levels of shedding, especially in vaginal mucus and faeces, the characteristics of the bacterium in the environment (i.e. its survival and the fraction of bacteria shed reaching the environment), and some physiological parameters related to the intermittency of shedding (transition probability from a non-shedding infected state to a shedding state) or to the transition from one type of shedder to another one (transition probability from a seronegative shedding state to a seropositive shedding state).Our study is crucial for the understanding of the dynamics of C. burnetii infection and optimization of control measures. Indeed, as control measures should impact the parameters influencing the bacterium spread most, our model can now be used to assess the effectiveness of different control strategies of Q fever within dairy cattle herds.     

Phage Therapy To Reduce Preprocessing Salmonella Infections in Market-Weight Swine

Contamination of meat products with food-borne pathogens usually results from the carcass coming in contact with the feces of an infected animal during processing. In the case of Salmonella, pigs can become colonized with the organism during transport and lairage from contaminated trailers and holding pens, resulting in increased pathogen shedding just prior to processing. Increased shedding, in turn, amplifies the likelihood of carcass contamination by magnifying the amount of bacteria that enters the processing facility. We conducted a series of experiments to test whether phage therapy could limit Salmonella infections at this crucial period. In a preliminary experiment done with small pigs (3 to 4 weeks old; 30 to 40 lb), administration of an anti-Salmonella phage cocktail at the time of inoculation with Salmonella enterica serovar Typhimurium reduced Salmonella colonization by 99.0 to 99.9% (2- to 3-log reduction) in the tonsils, ileum, and cecum. To test the efficacy of phage therapy in a production-like setting, we inoculated four market-weight pigs (in three replicates) with Salmonella enterica serovar Typhimurium and allowed the challenged pigs to contaminate a holding pen for 48 h. Sixteen naïve pigs were randomly split into two groups which received either the anti-Salmonella phage cocktail or a mock treatment. Both groups of pigs were comingled with the challenged pigs in the contaminated pen. Treatment with the anti-Salmonella phage cocktail significantly reduced cecal Salmonella concentrations (95%; P < 0.05) while also reducing (numerically) ileal Salmonella concentrations (90%; P = 0.06). Additional in vitro studies showed that the phage cocktail was also lytic against several non-Typhimurium serovars.The U.S. Centers for Disease Control and Prevention report approximately 40,000 culture-confirmed cases of salmonellosis each year in the United States, which result in approximately 400 deaths (5). Many Salmonella outbreaks are associated with meat and poultry (20), with contamination usually resulting from the carcass coming into contact with the feces of a Salmonella-infected animal during processing (22).There is an association between pork products and Salmonella, as swine are generally considered to be the second largest reservoir of the organism among food animals after poultry. Although infections in adult swine are normally asymptomatic, once colonized, pigs can shed the organism in the feces for weeks and sometimes months (7).While a great deal of research has been done on developing on-farm anti-Salmonella intervention strategies, these methods are confounded by the fact that Salmonella prevalence in pigs often increases once the animals leave the farm as a result of (i) stress-induced reactivation of preexisting infections (14), (ii) new infections from contaminated transport trailers and processing facility holding pens (12, 15, 24, 31), or (iii) both. Consequently, animals with no history of previous Salmonella infection can begin shedding the organism just prior to processing, which is highly problematic in terms of food safety.We hypothesized that phage therapy could be developed as an effective means to counteract transport- and lairage-associated increases in Salmonella colonization in swine. Phage therapy has the advantage of being natural, nontoxic, and relatively inexpensive and could be used just prior to slaughter, unlike many antibiotics (18, 28). Here we describe a series of experiments demonstrating that treating market-weight pigs with an anti-Salmonella phage cocktail prior to their comingling with Salmonella-infected pigs in a highly contaminated environment resulted in reductions in Salmonella colonization. We further show that the phage cocktail could be effectively microencapsulated, making feed or water delivery possible.     

Using In Vitro Immunomodulatory Properties of Lactic Acid Bacteria for Selection of Probiotics against Salmonella Infection in Broiler Chicks

Poultry is known to be a major reservoir of Salmonella. The use of lactic acid bacteria has become one of successful strategies to control Salmonella in poultry. The purpose of this study was to select lactic acid bacteria strains by their in vitro immunomodulatory properties for potential use as probiotics against Salmonella infection in broiler chicks. Among 101 isolated lactic acid bacteria strains, 13 strains effectively survived under acidic (pH 2.5) and bile salt (ranging from 0.1% to 1.0%) conditions, effectively inhibited growth of 6 pathogens, and adhered to Caco-2 cells. However, their in vitro immunomodulatory activities differed significantly. Finally, three strains with higher in vitro immunomodulatory properties (Lactobacillus plantarum PZ01, Lactobacillus salivarius JM32 and Pediococcus acidilactici JH231) and three strains with lower in vitro immunomodulatory activities (Enterococcus faecium JS11, Lactobacillus salivarius JK22 and Lactobacillus salivarius JM2A1) were compared for their inhibitory effects on Salmonella adhesion and invasion to Caco-2 cells in vitro and their antimicrobial effects in vivo. The former three strains inhibited Salmonella adhesion and invasion to Caco-2 cells in vitro, reduced the number of Salmonella in intestinal content, spleen and liver, reduced the levels of lipopolysaccharide-induced TNF-α factor (LITAF), IL-1β, IL-6 and IL-12 in serum and increased the level of IL-10 in serum during a challenge study in vivo more efficiently than the latter three strains. These results suggest that in vitro immunomodulatory activities could be used as additional parameters to select more effective probiotics as feed supplements for poultry.     

The Effects of Vaccination and Immunity on Bacterial Infection Dynamics In Vivo

Salmonella enterica infections are a significant global health issue, and development of vaccines against these bacteria requires an improved understanding of how vaccination affects the growth and spread of the bacteria within the host. We have combined in vivo tracking of molecularly tagged bacterial subpopulations with mathematical modelling to gain a novel insight into how different classes of vaccines and branches of the immune response protect against secondary Salmonella enterica infections of the mouse. We have found that a live Salmonella vaccine significantly reduced bacteraemia during a secondary challenge and restrained inter-organ spread of the bacteria in the systemic organs. Further, fitting mechanistic models to the data indicated that live vaccine immunisation enhanced both the bacterial killing in the very early stages of the infection and bacteriostatic control over the first day post-challenge. T-cell immunity induced by this vaccine is not necessary for the enhanced bacteriostasis but is required for subsequent bactericidal clearance of Salmonella in the blood and tissues. Conversely, a non-living vaccine while able to enhance initial blood clearance and killing of virulent secondary challenge bacteria, was unable to alter the subsequent bacterial growth rate in the systemic organs, did not prevent the resurgence of extensive bacteraemia and failed to control the spread of the bacteria in the body.     

A Five-Strain Probiotic Combination Reduces Pathogen Shedding and Alleviates Disease Signs in Pigs Challenged with Salmonella enterica Serovar Typhimurium

Salmonella spp. infection is a major cause of gastroenteritis, with many thousands of cases reported in the European Union every year. The use of probiotics offers the potential to improve this situation. Here, we investigate the effects of oral treatment of pigs with a defined lactic acid bacteria culture mixture on both clinical and microbiological signs of Salmonella enterica serovar Typhimurium infection. Fifteen weaned pigs blocked by sex and weight were administered control milk or a mixture of five probiotic strains as either a milk fermentate or milk suspension for a total of 30 days. The mixture consisted of two strains of Lactobacillus murinus and one strain each of Lactobacillus salivarius subsp. salivarius, Lactobacillus pentosus, and Pediococcus pentosaceous. Following probiotic administration for 6 days, animals were challenged orally with serovar Typhimurium; the health of the animals and the microbiological composition of their feces were monitored for 23 days postinfection. Animals treated with probiotic showed reduced incidence, severity, and duration of diarrhea. These animals also gained weight at a greater rate than control pigs administered skim milk. Mean fecal numbers of Salmonella were significantly reduced in probiotic-treated animals at 15 days postinfection (P = 0.01). The administered probiotic bacteria improved both the clinical and microbiological outcome of Salmonella infection. These strains offer significant benefit for use in the food industry and may have potential in human applications.     

Fecal Shedding of Zoonotic Food-Borne Pathogens by Wild Rodents in a Major Agricultural Region of the Central California Coast

Recent outbreaks of food-borne illness associated with the consumption of produce have increased concern over wildlife reservoirs of food-borne pathogens. Wild rodents are ubiquitous, and those living close to agricultural farms may pose a food safety risk should they shed zoonotic microorganisms in their feces near or on agricultural commodities. Fecal samples from wild rodents trapped on 13 agricultural farms (9 produce, 3 cow-calf operations, and 1 beef cattle feedlot) in Monterey and San Benito Counties, CA, were screened to determine the prevalence and risk factors for shedding of several food-borne pathogens. Deer mice (Peromyscus maniculatus) were the most abundant rodent species trapped (72.5%). Cryptosporidium species (26.0%) and Giardia species (24.2%) were the predominant isolates from rodent feces, followed by Salmonella enterica serovars (2.9%) and Escherichia coli O157:H7 (0.2%). Rodent trap success was significantly associated with detection of Salmonella in rodent feces, while farm type was associated with fecal shedding of Cryptosporidium and Giardia. Seasonal shedding patterns were evident, with rodents trapped during the spring and summer months being significantly less likely to be shedding Cryptosporidium oocysts than those trapped during autumn. Higher rodent species diversity tended to correlate with lower fecal microbial prevalence, and most spatiotemporal pathogen clusters involved deer mice. Rodents in the study area posed a minimal risk as environmental reservoirs of E. coli O157:H7, but they may play a role in environmental dissemination of Salmonella and protozoa. Rodent control efforts that potentially reduce biodiversity may increase pathogen shedding, possibly through promotion of intraspecific microbial transmission.     

Are Salmonella-Induced Gastroenteritis Neglected in Developing Countries? Feedback from Microbiological Investigations in N’Djamena Hospitals,Chad

Salmonella is considered to be one of the main pathogens causing human gastroenteritis worldwide. Looking for Salmonella in Africa in patients suffering from gastroenteritis is rather unusual, and the use of antibiotics is not subject to any regulation. This study intends for stressing the possible prominent importance of Salmonella in digestive diseases in Africa as well as identifying antimicrobial resistance of Salmonella isolates from faeces samples of human origin. All samples were collected from five N’Djamena hospitals, from patients suffering from diarrhoea. The collecting was undertaken over two periods of six months each: from August 2010 to January 2011 and from September 2011 to February 2012. Salmonella isolates were obtained by standard cultivation and serotyping methods. A total of 43 Salmonella isolates were identified, belonging to 21 different serovars. The most prevalent serovar was Salmonella Stanleyville (n = 7), followed by S. Anatum (n = 4) and S. Kottbus (n = 3). The other serovars were under-represented. The majority of these isolates were susceptible to all antibiotics tested (CLSI Standards), except two S. Enteritidis isolates that exhibited resistance to fluoroquinolones. The different serovars and antibiotic resistance profiles that were observed highlight the substantial diversity of Salmonella in N’Djamena, Chad. Roughly, one out of ten patients who consulted for gastroenteritis was shedding Salmonella spp. and none of them would have been diagnosed outside the context of this research program. This study may encourage local clinicians to explore more often salmonellosis suspicion in their daily practice.     

Are we overestimating risk of enteric pathogen spillover from wild birds to humans?

Enteric illnesses remain the second largest source of communicable diseases worldwide, and wild birds are suspected sources for human infection. This has led to efforts to reduce pathogen spillover through deterrence of wildlife and removal of wildlife habitat, particularly within farming systems, which can compromise conservation efforts and the ecosystem services wild birds provide. Further, Salmonella spp. are a significant cause of avian mortality, leading to additional conservation concerns. Despite numerous studies of enteric bacteria in wild birds and policies to discourage birds from food systems, we lack a comprehensive understanding of wild bird involvement in transmission of enteric bacteria to humans. Here, we propose a framework for understanding spillover of enteric pathogens from wild birds to humans, which includes pathogen acquisition, reservoir competence and bacterial shedding, contact with people and food, and pathogen survival in the environment. We place the literature into this framework to identify important knowledge gaps. Second, we conduct a meta‐analysis of prevalence data for three human enteric pathogens, Campylobacter spp., E. coli, and Salmonella spp., in 431 North American breeding bird species. Our literature review revealed that only 3% of studies addressed the complete system of pathogen transmission. In our meta‐analysis, we found a Campylobacter spp. prevalence of 27% across wild birds, while prevalence estimates of pathogenic E. coli (20%) and Salmonella spp. (6.4%) were lower. There was significant bias in which bird species have been tested, with most studies focusing on a small number of taxa that are common near people (e.g. European starlings Sturnus vulgaris and rock pigeons Columba livia) or commonly in contact with human waste (e.g. gulls). No pathogen prevalence data were available for 65% of North American breeding bird species, including many commonly in contact with humans (e.g. black‐billed magpie Pica hudsonia and great blue heron Ardea herodias), and our metadata suggest that some under‐studied species, taxonomic groups, and guilds may represent equivalent or greater risk to human infection than heavily studied species. We conclude that current data do not provide sufficient information to determine the likelihood of enteric pathogen spillover from wild birds to humans and thus preclude management solutions. The primary focus in the literature on pathogen prevalence likely overestimates the probability of enteric pathogen spillover from wild birds to humans because a pathogen must survive long enough at an infectious dose and be a strain that is able to colonize humans to cause infection. We propose that future research should focus on the large number of under‐studied species commonly in contact with people and food production and demonstrate shedding of bacterial strains pathogenic to humans into the environment where people may contact them. Finally, studies assessing the duration and intensity of bacterial shedding and survival of bacteria in the environment in bird faeces will help provide crucial missing information necessary to calculate spillover probability. Addressing these essential knowledge gaps will support policy to reduce enteric pathogen spillover to humans and enhance bird conservation efforts that are currently undermined by unsupported fears of pathogen spillover from wild birds.     

Enhancement of Immunohistochemical Detection of Salmonella in Tissues of Experimentally Infected Pigs

Salmonella Typhimurium is one of the main pathogens compromising porcine and human health as well as food safety, because it is a prevailing source of foodborne infections due to contaminated pork. A prominent problem in the management of this bacteriosis is the number of subclinically infected carrier pigs. As very little is known concerning the mechanisms allowing Salmonella to persist in pigs, the objective of this study was to develop an immunohistochemical approach for the detection of salmonellae in tissue of pigs experimentally infected with Salmonella Typhimurium. Samples were obtained from a challenge trial in which piglets of the German Landrace were intragastrically infected with Salmonella enterica serovar Typhimurium DT104 (1.4-2.1×10¹⁰ CFU). Piglets were sacrificed on days 2 and 28 post infection. Tissue samples of jejunum, ileum, colon, ileocecal mesenteric lymph nodes (Lnn. ileocolici), and tonsils (Tonsilla veli palatini) were fixed in Zamboni’s fixative and paraffin-embedded. Different immunohistochemical staining protocols were evaluated. Salmonella was detected in varying amounts in the tissues. Brown iron-containing pigments in the lymph nodes interfered with the identification of Salmonella if DAB was used as a staining reagent. Detergents like Triton X-100 or Saponin enhanced the sensitivity. It seems advisable not to use a detection system with brown staining for bacteria in an experimental setup involving intestinal damage including haemorrhage. The use of detergents appears to result in a higher sensitivity in the immunohistochemical detection of salmonellae.Key words: Swine, immunohistochemistry, histochemistry, bacteria, detection limit