Brian W. SHELDON
Department Extension Leader & Professor
Department of Poultry Science,
North Carolina State University
Raleigh, NC
U.S.A.
Introduction
The focus of this presentation will be to review the relevant literature on the impact of layer molting practices on Salmonella contamination and shedding and to summarize the findings of a commercial molting study recently conducted at North Carolina State University. The objectives of this study were (i) to determine whether the hen’s laying cycle and use of a reduced energy, protein and calcium molting diet influenced the prevalence and populations of Salmonella found in layer feces, and (ii) to characterize Salmonella fecal isolates from a commercial laying hen complex by serotype, pulsed field gel electrophoresis (PFGE) and antibiotic susceptibility.
The importance of this topic is related to the fact that non-typhoid salmonellosis remains a serious public health concern in the United States. An estimated 1.4 million cases of Salmonella-associated gastroenteritis are diagnosed annually in the United States (Mead, 1999). These illnesses result in considerable hospitalizations and around 600 deaths. Estimated costs for medication and losses in productivity stemming from these illnesses range from $372 million to $4 billion annually (Roberts, 1988; Angulo and Swerdlow, 1998). Raw meat and poultry are recognized as the primary sources for transmitting Salmonella species to humans, with 40% of the clinical cases attributed to the consumption of egg and poultry products (Sanchez et al., 2002). Infection of the reproductive organs of laying hens often is the underlying factor leading to the production of contaminated eggs.
Animal welfare advocates have been quite critical of the shell egg industry over the use of caged layer systems and their feed withdrawal molting practices that reportedly place the layers under excessive stress and increase their susceptibility to Salmonella infection. For these reasons it is very important to fully evaluate the impact of the hen’s egg production cycle and molting practices on the dynamics of Salmonella species populations and prevalence. The general purpose of induced molting of commercial layers is to rejuvenate the hen’s reproduction system so that better eggshell quality and higher egg production can be achieved and extended over several laying cycles. An induced molt has historically been accomplished by fasting or complete withdrawal of feed from the flock for a specified time period or by limiting energy and critical nutrient dietary inputs such protein, calcium, and potassium until or beyond the time where egg production ceases. Molting programs in the U.S. involve an estimated 75-80% of the commercial flocks and 100% of California’s flocks. At any given point in time, 25-30% of the nation’s layers are either in a molt or have been molted earlier (Bell, 2001). Considering that there is an estimated 256 million hens in the U.S., the number of hens molted annually would be between192 to 204 million (Holt, 2003).
It has been noted that feed withdrawal from hens, while effective in inducing a molt and allowing an adequate reproductive rest period for the hen, may cause deleterious effects on the animal such as greater intestinal colonization by Salmonella enterica subspecies enterica serovar Enteritidis (SE), one of the leading causes of salmonellosis in humans. There are several physiological effects that result from molting layers including a decline in body weight; a reduction in the size of the ovaries, oviduct, and liver; elevated serum packed blood volume, haemoglobin levels and corticosterone and thyroid hormones; and declining plasma gonadotropins and sexual steroids (Holt, 1999).
Although feed removal provides the benefit of extending the effective egg-laying life of the flock, it may cause some undesirable effects on the bird’s immune system such as reduced humoral and cell-mediated immunity. The consequence of an impaired immune response may allow invasion by pathogenic microorganisms residing in the hen’s environment (Holt, 1999). The discovery that the immune system of molted hens was compromised prompted investigations into the effect of the practice on infection by Salmonella. Indeed, hens experimentally challenged with an exogenous source of SE concomitantly during molt induction exhibited a much more severe infection as compared to unmolted hens. The rates of intestinal shedding seen in molted birds were reportedly higher (Holt and Porter, 1992). Moreover, the molted hens also shed higher populations of SE over that of the control hens (Holt, 1992; Holt, 1993; Holt et al., 1994). Molted hens, unlike their control counterparts who are generally asymptomatic towards the presence of Salmonella, exhibited inflamed intestines, primarily in the colon and cecum (Holt and Porter, 1992). Feed withdrawal to induce a molt can also lead to the recurrence of a previous SE infection with significantly higher rates of shedding and larger populations (Holt and Porter, 1993). In their study, Holt and Porter found that experimentally-SE infected molted hens transmitted the organism horizontally to 85-100% (in contrast to 10-30% of the unmolted birds) of previously uninfected, but contact-exposed, hens (i.e., control birds located in adjacent cages) after 10 days following the initial SE challenge of the hens. Less dramatic effects were detected when hens were dosed with lower populations of SE. The molted hens also produced more eggs that were contaminated with SE. These findings suggest that molting can influence an SE infection at different times in the infection cycle and that rapid transmission of SE can occur over significant distances between infected and molted hens to unmolted birds indicating a possible airborne route of transmission.
The exacerbation of infection and ready transmission of SE to previously uninfected hens indicate that the molted hens may be more susceptible to infection by SE. This in fact was experimentally proven by Holt (1993) and Holt and co-investigators (1994). They found that it took only 10 SE organisms to infect 50% of the molted hens in contrast to 103-104 SE organisms to infect a test group of unmolted hens. The molted hens were acutely susceptible to SE infection, which could put them at greater risk for any SE found in their environment.
The discovery that molting through a withdrawal program exacerbates an SE infection in hens prompted research into methods to ameliorate the situation through such measures as altering methods for inducing the molt or through other intervention strategies. In one example, a low energy, low calcium diet which was previously shown to pause egg production in hens (Rolon et al., 1993) was compared to a feed withdrawal molt as to their relative impact on SE infection. The SE shed rate between the two molting programs was similar. However, birds fed the molt diet shed fewer SE organisms, were less susceptible to SE infections, and exhibited less inflammation of the intestine (Holt et al., 1994). These findings indicate that producers may be able to recycle their aging flocks without completely withdrawing feed. In 1997 Corrier et al. administered lactose in the drinking water during a hen molt which caused a reduction in the cecal carriage and extraintestinal dissemination of an SE challenge. Other treatment strategies that have been evaluated under experimental conditions to ameliorate SE challenges during molting or to eliminate the need for complete feed withdrawal include the use of dietary alfalfa (Landers et al., 2005) and feed processing by-products (i.e., wheat middlings, cottonseed meals and jojoba meal), antibiotic therapy, and vaccination with killed or live attenuated S. Enteritidis or Typhimurium vaccines. Each of these approaches provides the producer with many potential solutions to the SE food safety issue yet still allows the producer to recycle their hens. Unfortunately many of the past and current studies involving these and other alternatives have been evaluated under experimental conditions and thus do not necessarily translate to what is occurring under on-farm commercial conditions. Some field studies such as the S. Enteritidis Pilot Project in Pennsylvania (U.S. Department of Agriculture, 1995) tell the story as to the efficacy of these alternative molting strategies to ameliorate the hen’s heightened susceptibility to SE infection.
As indicated above, a considerable number of experimental research studies have been conducted over the past 5 to 10 years to determine the prevalence of Salmonella on layer farms, yet few have actually identified and enumerated Salmonella populations. Thus, the diversity of Salmonella strains found in commercial layer flocks remains largely unknown. Merely monitoring for the presence or absence of Salmonella does not provide adequate information to adequately assess the true risks of table eggs in contributing to human disease. Another important and controversial practice has been the practice of using sub therapeutic and therapeutic use of antibiotics in commercial poultry production operations for controlling disease and improving the growth and efficiency of feed conversion (Levy, 1987; Franco et al., 1990). The management practices along with the use of medicated feeds in broiler and layer operations has been suggested to result in the shedding of antibiotic resistant strains of Salmonella into the poultry environment (D’Aoust, et al., 1992). Knowledge of the prevalence, populations and characteristics of Salmonella isolates from farm animal wastes are important to understand when establishing animal waste management programs and for evaluating and preventing the risk of transmitting this pathogen to ground and surface waters and subsequently to humans.
Materials and methods of North Carolina State University study
Study site and sample collection. This study was conducted between September 2003 and August 2004 at a commercial North Carolina layer facility containing 12 high-rise houses. Each house measured 57 x 480 feet and housed around 77,000 egg-laying Leghorn hens in cages arranged across 6 rows. Temperatures in the layer houses were typically controlled between 20 and 28ºC. Temperatures and ammonium concentrations in the houses were measured during each visit to the farm for taking samples. According to the producer all pullets were initially vaccinated on the pullet farm for Salmonella Typhimurium and at 18 wk of age were transferred to the layer farm over the course of two to three days. All flocks were molted between 66 and 74 wk using a non-fasting molt program comprised of adjusting the protein, calcium and energy dietary concentrations and lighting program (8hr light:16hr dark) as prescribed under the United Egg Producers Animal Welfare Guidelines. Over the course of the 8-week molt period the birds received four diets, which provided for initiation of the molt (i.e., cessation of egg production) followed by a series of resting diets to allow for rejuvenation of the body stores. On each sampling day approximately 300 grams of fresh excreta were collected from beneath each row of cages that had been retrofitted with capture panels and pooled together by rows into individual sterile WhirlPak bags. The samples were kept in coolers containing ice packs, transported to the laboratory (within two hours) and then processed on the same day to determine Salmonella prevalence and populations.
Salmonella identification and enumeration. A most probable number (MPN) procedure was employed to estimate Salmonella populations contained in the layer excreta. Upon arrival at the laboratory the WhirlPak bags containing the fresh excreta were thoroughly mixed by hand to yield a homogeneous mixture. Twenty-five grams of each composite sample were placed into a sterile stomacher filter bag containing 50 ml of buffered peptone water (BPW; Oxoid, Ogdensburg, NY). Individual bags were stomached for one minute. For pre-enrichment, the mixtures were serially diluted in BPW, incubated at 37ºC for 18 to 24 h (Moringo et al., 1986; Tate and Miller, 1990; Tate et al., 1992), and then 0.1 ml of the appropriate dilutions from each tube transferred to three tubes containing 10 ml of Rappaport Vassiliadis (RV) broth (Oxoid, Ogdensburg, NY). All RV tubes were incubated at 42ºC for 18 to 24 h for selective enrichment of Salmonella species. Following incubation, one 10 µl loopful from each RV tube was streaked onto modified lysine iron agar (MLIA; selective medium for isolating Salmonella, Oxoid) and incubated at 37ºC for 18 to 24 h. Suspect black colonies on MLIA plates were picked and confirmed for Salmonella by biochemical tests, reaction on triple sugar iron agar slants (TSI; Oxoid) and agglutination using Salmonella poly-O antiserum (Difco Laboratories, Detroit, MI). Populations of Salmonella species were calculated using the Thomas’ approximation (Swanson et al., 2001) of MPN/g = P/(NT)1/2 where P = the number of positive tubes, N = the total quantity of sample (g) in all negative tubes, and T = the total quantity of sample (g) in all tubes. The minimum detection limit for this method was 10 organisms per gram of excreta (one log).
In order to increase detection sensitivity, the prevalence of Salmonella in the excreta was tested as follows. Each 25 gram sample was placed into a sterile stomacher filter bag containing 100 ml of BPW. The bags were stomached for one minute and then an additional 125 ml of BPW was added, mixed thoroughly, and incubated at 37ºC for 18 to 24 h. One ml from each bag was added to a bottle containing 100 ml of RV broth. All bottles were incubated at 42ºC for 18 to 24 h. The remaining procedures for isolating and identifying Salmonella were as described above.
Serotyping. Serotyping is a useful classification scheme for tracking trends in Salmonella surveillance data over time. There are currently around 2,449 known serotypes of Salmonella (Tauxe, 1991). The Kauffmann-White serotyping scheme detects an antigenic variation in the lipopolysaccharide (O-antigen) and flagella (H-antigen) and allows for the typing of over 2,100 different Salmonella serovars (Edwards et al., 2002). A total of 45 Salmonella isolates recovered from the laying hen faeces (17 isolates from 18-wk birds, 17 isolates from 25-28-wk birds, 7 isolates from 66-74-wk birds and 4 isolates from 75-76-wk birds) were selected for serotyping. For serotyping, the Salmonella isolates were transferred onto tryptic soy agar (Difco, Becton Dickinson) slants, grown overnight at 37ºC and shipped by overnight courier to the USDA National Veterinary Service Laboratories in Ames, Iowa.
Antibiotic resistance analysis. After being serotyped, the susceptibility of the 45 Salmonella isolates to a battery of antibiotics was determined using the disk diffusion method described by the National Committee for Clinical Laboratory Standards (NCCLS, 2000). The selection of antibiotics for testing was based on the recommendations of the NCCLS. The susceptibility tests were conducted using the Sensititre Susceptibility System (Sensititre®, TREK Diagnostic Systems Inc, Cleveland, OH). The system encompasses a microtitre plate that is dosed with 15 individual antimicrobial agents at specified concentrations and then dried. The names of the 15 antibiotics, their concentrations, and the minimum inhibitory concentration break points are listed in Table 1. A standard protocol as described under the National Antimicrobial Resistance Monitoring System (NARMS) was followed to evaluate antibiotic susceptibility of the 45 Salmonella isolates. Each isolate was cultured on BHI agar plates at 37ºC for 18 to 24 hours. One to two colonies from the BHI plate were transferred to 5 ml of sterile saline (0.9% NaCl) and adjusted to 0.5 McFarland (ca. 1 x 108 CFU/ml) using a nephelometer (Promega, Madison, WI) calibrated using a 0.5 McFarland barium sulphate turbidity standard (Sensititre®, TREK Diagnostic Systems, Inc). Seventy-five µl of the saline suspension were transferred to 10 ml of Mueller Hinton (MH) broth (Oxoid) and 50 µl of the MH broth suspension transferred to each of the microtitre plate wells. The plates were then sealed with an adhesive seal and incubated at 37ºC for 18 to 24 h. The plates were manually read for bacterial growth using a microtiter plate holder positioned under a florescent lamp. A Salmonella Typhimurium DT104 (ATCC 700408) strain served as the quality control standard for the assays.
Pulsed field gel electrophoresis (PFGE). The procedures for extracting Salmonella genomic DNA and conditions for running PFGE were as described in the CDC-PulNet standard method with minor modifications. In brief, a single Salmonella colony was picked, inoculated into 10 ml of BHI broth (Oxoid, Ogdensburg, NY) and incubated at 37ºC for 16 to 18 h in a shaking water bath (200 rpm). One milliliter aliquots of the cultures were pelleted by centrifugation, washed three times in phosphate buffered saline (PBS), and then the optical density (at 600 nm) adjusted to 0.10. One ml of this cell suspension was added to 25 µl of proteinase K (Qiagen, 20 mg/ml) and mixed gently by inversion. Chromosome grade agarose (Bio-Rad Laboratories, Hercules, CA) was prepared in 1x TE buffer (10 mM Tris, 1 mM EDTA) to a final concentration of 2% and tempered at 55°C. Plugs were formed by mixing 0.5 ml of the cell suspension with 0.5 ml of 2% agarose and then transferred to disposable gel plug molds (Bio-Rad Laboratories). After solidification the plugs were transferred to lysis buffer (75 mM Tris, pH 8.0; 75 mM EDTA, pH 8.0; 1.5% sarcosine; 0.25 mg of proteinase K per ml) and incubated at 54°C overnight. The lysis buffer was removed and then the plugs were first washed for 15 min in 5 ml of sterile prewarmed distilled water at 54°C followed by 20 min in 5 ml of TE. A final set of four washes of 20 min duration each was completed using 5 ml of TE buffer per wash. Plugs were then cut to 2 mm widths using a scalpel and them transferred to small eppendorf tubes containing 200 µl of 1x XbaI buffer (Promega) at room temperature (RT). After 30 min the buffer was removed and replaced with a fresh mixture containing 50 U of XbaI restriction enzyme in 1x restriction buffer and incubated overnight at 37ºC. The plugs were subsequently soaked in standard 0.5x Tri-borate-EDTA (TBE, Promega) prior to electrophoresis (at least 30 min at RT). Restriction fragments of the DNA were separated using the CHEF DRIII system (Bio-Rad Laboratories, Hercules, CA) and 1% PFGE agarose (Bio-Rad Laboratories) with 2.2 liters of standard 0.5x TBE buffer. The electrophoretic conditions were as follows: initial switch time, 6 s; final switch time, 40 s; gradient, 6V/cm; included angle, 120; run time, 22 h; cooling temperature, 14°C. After electrophoresis the gels were stained for 20 min in ethidium bromide (0.01%) staining solution, destained with distilled water for 20 min, the water replaced and then destained for another 20 min. Gel images were digitally captured using a Pharmacia ImageMaster system. Gels were analyzed by visual interpretation. PFGE profiles differing in one or more bands were considered different.
Statistical analysis. The composited fresh faecal samples collected from beneath each row of cages served as an independent trial replicate. There were 18, 24, 18 and 18 sample replicates evaluated for the 18 wk (pullet placement), 25 to 28 wk (peak of 1st production cycle), 66 to 74 wk (molting) and 75 to 76 wk (peak of 2nd production cycle) old birds, respectively. The independent variables were bird ages, house temperatures and ammonia levels. The data were analyzed by the general linear model (GLM) procedure for analysis of variance (ANOVA). When a significant effect was observed means were compared using the Pdiff option of SAS (SAS, 1990). Model and parameter adequacy was considered significant at P ≤ 0.05 unless otherwise noted.
Results and Discussion
Layer house ammonia levels and temperatures. The house ammonia levels and temperatures for different ages of laying hens are summarized in Table 2. The ammonia level in houses containing the 25-28 wk hens was 55.0 ppm which was significantly higher (P < 0.001) than the 18 wk (15.0 ppm) and 75-76 wk old birds (15.6 ppm). The houses containing the molted birds had the lowest ammonium levels averaging 5.8 ppm. In contrast to ammonia concentrations, there were no differences (P > 0.001) in house temperatures across all four age categories (range between 24.2 and 26.3°C). House management practices (ventilation, sanitation program, pest control, mortality removal, temperature controls, etc.) were consistently applied across all layer houses. Ammonia in poultry house is generated mainly from the microbial decomposition of uric acid in the manure. Microbial uricase in poultry manure is an important key enzyme for reducing ammonia production. The higher ammonia levels detected in the layer houses containing 25-28 wk hens is related to the bird’s higher metabolism, greater feed and higher protein intake, and subsequent production of larger amounts of manure. Ammonia is typically considered an indoor air quality concern for livestock and poultry producers since elevated levels can negatively impact animal health and production. For example, reduced final body weights were observed in poultry reared in houses having indoor ammonia levels of 25 ppm or higher during the brooding stage (Reece et al., 1980).
Salmonella populations and prevalence. Salmonella populations and prevalence in manure samples taken from the four hen age groups are summarized in Table 3. For the 18-wk birds, the Salmonella populations ranged from 1.00 to 1.97 log MPN/g with a mean value of 1.25 log MPN/g. For the 25-28 wk birds, the populations ranged from 1.00 to 2.73 log MPN/g with a mean value of 1.27 log MPN/g. For the 66-74 and 75-76 wk birds, the Salmonella populations ranged from 1.00 to 3.76 log MPN/g (mean of 1.20 log MPN/g) and 1.00 to 1.89 log MPN/g (mean of 1.14 log MPN/g), respectively. No significant differences (P > 0.05) in Salmonella populations were detected among the four age groups. However, there were substantial differences in the prevalence of Salmonella detected among these groups. The 18-week hens had the highest prevalence of Salmonella (55.6%) and the lowest mean house ammonia concentrations (15.0 ppm). In contrast, the 25-28 wk hens had a lower Salmonella prevalence rate (41.7%) and the highest ammonia levels (55 ppm). The molted hens yielded the lowest Salmonella prevalence rate of 5.5% and a low ammonia concentration (5.8 ppm). These findings indicate that the non-fasting molting program employed by this commercial producer did not exacerbate Salmonella shedding. In contrast to these findings, laying hens that were fasted (feed withdrawn) and experimentally infected with 106 Salmonella Enteritidis (SE) organisms yielded the highest level of faecal shedding during molting (Holt, 1993; Holt and Porter, 1992 and 1993; Holt et al., 1994). These previous studies by Holt and colleagues documented that under experimental and controlled conditions, induced molting via feed removal significantly depressed the cell-immune response of layers and increased the hen’s susceptibility to Salmonella contamination (Holt, 1992; Holt, 1999). In comparison, Holt (2003) found that the use of low-energy, low-calcium diets versus complete feed withdrawal significantly decreased Salmonella shedding during the molting process.
Many sources of Salmonella contamination are possible on poultry farms including pullets, air, feed, water, flies, rodents, and humans (Hoover et al., 1997; Craven et al., 2000; Heyndrickx et al., 2002; Liljebjelke et al., 2005). For the commercial farm evaluated in this study we observed numerous flies outside the houses yet few on the inside. Moreover, on several occasions we observed some dead mice inside the houses. In order to improve the understanding of the ecology and diversity of Salmonella isolates on this layer facility, we further characterized the recovered faecal Salmonella isolates by serotype, PFGE and antibiotic resistance analysis.
Characterization of Salmonella isolates. The Salmonella serovars identified across the four bird age groups are summarized in Table 4. Eight serotypes were identified: Salmonella Kentucky (62% of isolates), Montevideo (11%), Typhimurium (var. 5-, 4%), Heidelberg (4%), Senftenberg (2%), 8,(20): Nonmotile (2%), 8,(20):-:z6 (2%), and nontyped (11%). Salmonella Enteritidis was not detected from among the picked colonies. Salmonella Kentucky was the most prevalent serotype across all layer age groups. Salmonella surveillance studies conducted by the Centers for Disease Control and Prevention identified Salmonella enterica Typhimurium and Enteritidis as the most commonly reported serovars associated with human illness (CDC Salmonella annual summary, 2003); Salmonella Heidelberg, Montevideo and Senftenberg were also listed among the top 20 serovars identified in the 2003 report. In contrast to these human isolates, Salmonella Kentucky and Heidelberg were the top two serotypes identified from chickens and turkeys (U.S. Department of Agriculture, 1999). Approximately 50% of the isolates from chicken and turkey sources were Salmonella Kentucky. Many factors may influence why Salmonella Kentucky is more commonly isolated from poultry than from human clinical isolates. Several proposed explanations include variations in host specificity, dose response, and differences in cultural methods (Ordal et al., 1976; Juven et al., 1984; Chalker and Blaser, 1988; Mohammed and Hinton, 1993; Sarwari et al., 2001). In our study we discovered that when employing the official Bacteriological Analytical Method for detecting Salmonella (AOAC, 2003), the population of Salmonella Enteritidis was 100-fold lower than the populations of the other 9 Salmonella serovars: Kentucky, Heidelberg, Typhimurium, Senftenberg, Muenster, Hadar, Infantis, Mbandaka, and Newport. Due to the cost of serotyping, laboratories often pick just a single colony from among the numerous other colonies that may be present on an individual culture plate. If S. Enteritidis represents a smaller proportion of the total population of Salmonella serotypes, the probability of picking one S. Enteritidis colony would be considerably lower than for the other serotypes.
These forty-five identified Salmonella serovar isolates were further characterized by PFGE and antibiotic resistance profiles. The results are summarized in Tables 5 and 6, respectively. Thirty-five percent (16 of 45) of the Salmonella isolates were resistant to at least one antibiotic. There were 10 antibiotic resistance patterns detected including Tet, Strep, Strep/Tet, Amp/Tet/Ceftio, Amp/Cefox/Ceftio, Amp/Ceftio, Amp/Strep/Tet, Amp/Strep/Tet/Ceftio, Amp/Tet and Amp/Strep/Tet/Cefox/Ceftio. A large proportion of the Salmonella isolates were resistant to tetracycline, ampicillin, streptomycin and ceftiofur, all widely used in the treatment of human systemic salmonellosis cases (D’Aoust et al., 1992). No resistance to gentamicin, kanamycin and nalidixic acid was observed. Livestock and poultry producers rely on antibiotics to treat a host of diseases and infections. Such treatments help promote the animal’s health and well-being in addition to ensuring a safer food supply for consumers. The concern with using any antibiotic for either animal or human use is that residual levels of antibiotic remaining in the GI tract would be voided and thus may linger in the environment. This so-called pharmaceutical pollution can subsequently lead to bacterial mutations and the formation of antibiotic resistant strains of bacteria.
Among the 8 serotypes identified, Salmonella Kentucky, untypeable, Typhimurium (var. 5-), and Montevideo displayed some antibiotic resistance whereas Heidelberg, Senftenberg, 8,(20): Nonmotile and 8,(20):-:z6 showed no resistance. There were 5, 3 and 2 antibiotic resistant patterns observed for Salmonella Kentucky, untypeable, and Montevideo, respectively. Among the 16 Salmonella isolates that were found to be antibiotic resistant, 8 of the isolates were typed as S. Kentucky, 5 isolates were untypeable, 2 were Typhimurium, and 1 isolate was classified as Montevideo. The antibiotic resistant prevalence rates were 29 (8 of 28), 100 (5 of 5), 100 (2 of 2) and 20 (1 of 5) percent for Salmonella Kentucky, untypeable, Typhimurium and Montevideo, respectively. Salmonella Kentucky was the most prevalent serotype but was not the most antibiotic resistant serotype. Variations in the proportion of antibiotic resistant isolates were also detected across flock age groups; 18% (3 of 17) of the 18-wk old birds were antibiotic resistant, 47% (8 of 17) of the 25-28 wk old birds, 43% (3 of 7) of the 66-74 wk old birds, and 50% (2 of 4) of the 75-76 wk old layers (Table 6). Interestingly, it appears that the faeces from older birds produced a greater percentage of isolates that were antibiotic resistant.
Among the Salmonella isolates typed by PFGE, there were 9 different genotypes (Table 5 and Figure 1). Isolates were considered genetically indistinguishable when their restriction patterns had the same numbers of bands and the corresponding bands were of the same molecular size (Tenover et al., 1995). Salmonella Kentucky possessed 4 different PFGE types: D, F, G, and H. One of the Salmonella Kentucky strains shared the same PFGE type D with the Salmonella Heidelberg strain. Salmonella strains with similar fingerprint patterns can represent different serotypes because one or more nucleotide repeating units can alter transcription signals or change the reading frame of genes encoding for cell surface molecules that are used in differentiating serotypes. These findings demonstrate that PFGE analysis may be a useful tool for identifying strain diversity.
Conclusions
The findings from this study show that this particular commercial layer complex was contaminated with multiple Salmonella serovars of varying antibiotic resistance patterns and PFGE genotypes. Moreover, laying hen egg production stages influenced the prevalence of Salmonella recovered from the faeces. Moreover, the specific non-fasting molting program used by this producer did not increase the populations or rate of shedding of Salmonella into the faeces.
Among the eight Salmonella serovars identified, Salmonella Kentucky was the most common serotype (62%) recovered from the layer faeces. The high prevalence rate of ampicillin, tetracycline and streptomycin-resistant Salmonella strains detected in the layer faeces may be of some public health concern if the organisms were to spread to the environment and contaminate surface water.
References are available on request.
From Proceedings of the “Midwest Poultry Federation Convention”, St. Paul, Minnesota, U.S.A.
