Survey of Salmonella Contamination in Chicken Layer Farms in Three ...

1471 Journal of Food Protection, Vol. 77, No. 9, 2014, Pages 1471–1480 doi:10.4315/0362-028X.JFP-14-021 Copyright G, International Association for Food Protection

Survey of Salmonella Contamination in Chicken Layer Farms in Three Caribbean Countries ABIODUN ADESIYUN,1* LLOYD WEBB,2 LISA MUSAI,3 BOWEN LOUISON,4 GEORGE JOSEPH,5 ALVA STEWART-JOHNSON,1 SANNANDAN SAMLAL,1 AND SHELLY RODRIGO6 1School of Veterinary Medicine, Faculty of Medical Sciences, University of the West Indies, St. Augustine, Trinidad and Tobago; 2Institute of Public Health Studies, College of Veterinary Medicine, Nursing and Allied Health, Tuskegee University, Tuskegee, Alabama 36088, USA; and 3Poultry Surveillance Unit, Ministry of Food Production, Port of Spain, Trinidad and Tobago; 4Ministry of Agriculture, Forestry and Fisheries, Ministerial Complex, Tanteen, St. George’s Grenada; 5Veterinary and Livestock Services, Ministry of Agriculture, Food Production, Rural Development and Fisheries, Castries, St. Lucia; and 6Department of Public Health and Preventive Medicine, School of Medicine, St. George’s University, University Centre, Grenada, West Indies

MS 14-021: Received 14 January 2014/Accepted 7 March 2014

ABSTRACT This study was conducted to investigate the demography, management, and production practices on layer chicken farms in Trinidad and Tobago, Grenada, and St. Lucia and the frequency of risk factors for Salmonella infection. The frequency of isolation of Salmonella from the layer farm environment, eggs, feeds, hatchery, and imported day-old chicks was determined using standard methods. Of the eight risk factors (farm size, age group of layers, source of day-old chicks, vaccination, sanitation practices, biosecurity measures, presence of pests, and previous disease outbreaks) for Salmonella infection investigated, farm size was the only risk factor significantly associated (P ~ 0.031) with the prevalence of Salmonella; 77.8% of large farms were positive for this pathogen compared with 33.3 and 26.1% of medium and small farms, respectively. The overall isolation rate of Salmonella from 35 layer farms was 40.0%. Salmonella was isolated at a significantly higher rate (P , 0.05) from farm environments than from the cloacae. Only in Trinidad and Tobago did feeds (6.5% of samples) and pooled egg contents (12.5% of samples) yield Salmonella; however, all egg samples from hotels, hatcheries, and airports in this country were negative. Salmonella Anatum, Salmonella group C, and Salmonella Kentucky were the predominant serotypes in Trinidad and Tobago, Grenada, and St. Lucia, respectively. Although Salmonella infections were found in layer birds sampled, table eggs appear to pose minimal risk to consumers. However, the detection of Salmonella-contaminated farm environments and feeds cannot be ignored. Only 2.9% of the isolates belonged to Salmonella Enteritidis, a finding that may reflect the impact of changes in farm management and poultry production in the region.

Worldwide, table eggs are used in the preparation of numerous commercial and homemade products (7, 18). Salmonellosis resulting from the consumption of eggs or egg products has been reported in outbreaks globally, with both public health and economic implications (11, 19, 26, 30, 32). Rearing of layer birds in Salmonella-free farm environments and obtaining hatching eggs and day-old chicks free of Salmonella have been reported as imperative for preventing Salmonella from entering the human food chain (27, 31, 35). Salmonella can contaminate eggs by both vertical and horizontal transmission (14, 15, 27, 31). The frequency of isolation of Salmonella reported elsewhere has ranged from 4 to 12% in layer birds (3, 16), from 0.95 to 33.3% for layer farm environments or litters (6, 34), from 12.6 to 19.2% for imported day-old chicks (6, 31), from 0.35 to 1.24% for hatching eggs (6), and from 13 to 29% for hatcheries (13, 35). Rodents also have been documented as a potential source of Salmonella in layer farm environments (22, 23, 38). * Author for correspondence. Tel: 868-645-4481; Fax: 868-645-7428; E-mail: [email protected].

Although all serotypes of Salmonella can cause eggborne salmonellosis, Salmonella Enteritidis has emerged as the serotype most frequently associated globally with most human epidemics (25, 33, 39). Since 1989, Salmonella Enteritidis had emerged in the Caribbean as a pathogen of public health concern, causing sporadic cases and outbreaks of diarrhea in both local and tourist populations. In 1996, this serotype was the most frequent cause of salmonellosis in the Caribbean (21). Salmonellosis has been increasing in frequency since 1986 in Trinidad and Tobago followed by Jamaica and Barbados (8). Indar et al. (20) reported that the prevalence of Salmonella on layer farms in Trinidad and Tobago was significantly higher on egg shells (4.7%) than in egg contents (1.2%). Adesiyun et al. (2) found that Salmonella Enteritidis was the serotype of 58.3% of the Salmonella isolates from composite eggs (shells, content, or both) collected from layer farms and supermarkets in Trinidad. To date, no comprehensive surveys of Salmonella in table eggs have been conducted in the Caribbean subregion, and the impact that changes in farm management and poultry production systems in the Caribbean region would have on the distribution of Salmonella is unknown.

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Several Caribbean countries import table and hatching eggs from within and outside the region, and the risk to consumers of Salmonella in eggs and egg products is of concern. The current study was conducted to survey Salmonella in layer birds and their farm environments, feeds, table eggs, hatcheries, imported eggs, and day-old chicks in three countries. The risk factors for Salmonella contamination of farm environments and eggs and infection of layers were also investigated, and the serotypes of the Salmonella isolates recovered were determined. MATERIALS AND METHODS Study strategy. The project was designed to be performed in the poultry industry in the Caribbean subregion. The first phase of the study involved dissemination of an electronic questionnaire to each regional chief veterinary officer, and the second phase involved sampling for Salmonella in countries that consented to participate in the study. Questionnaire. Questionnaires were sent to the chief veterinary officers or their designates in 27 Caribbean countries to elicit information for 1999 to 2010 regarding foodborne and eggborne outbreaks and agents implicated; importation of eggs (table or hatching), egg products, and day-old chicks (country of origin); distribution of imported eggs (hatcheries, supermarkets, local markets, or hotels) or chicks (layer or broiler farms); microbial testing of table eggs (local or imported) and birds (layers or broilers); and pathogens isolated. Only 11 (40.7%) of the 27 countries returned completed questionnaires. Selection of countries for the study. After analysis of the questionnaire data, the sample collection phase was conducted in three countries that consented to participate: Trinidad and Tobago (7 September to 3 October 2011), Grenada (17 to 21 October 2011), and St. Lucia (14 to 18 November 2011). Logistical problems (permission to conduct study and availability of technical support to identify and visit poultry farms and other venues) associated with coordinating the project in the three countries made it impossible for simultaneous (time and duration) sampling in these three countries. Types, numbers, and sources of samples. Samples were collected from layer farms, hatcheries, ports of entry, and the kitchens of hotels. The sample sizes were determined for each country using the following formula (37): n~1:962 Pexp 1{Pexp =d 2 where n is the estimated number of samples, Pexp is the expected prevalence of Salmonella, and d is the precision level. Based on the questionnaire data, the farms were classified as small (less than 5,000 birds), medium (5,001 to 10,000 birds), and large (more than 10,000 birds). Overall, the questionnaire was administered to a total of 81 layer farms in Grenada (21 farms), Trinidad and Tobago (23 farms), and St. Lucia (37 farms), but samples were collected from only 35 (43.2%) of the farms. In each of the three countries studied, the following types of samples were collected, wherever possible: (i) On layer farms, cloacal swabs of laying birds (19 per small farm, 56 per medium farm, and 57 per large farm), drag samples of each pen in use (deep litter system), feeds in use at the time of farm visit, and freshly laid eggs (6 per small farm and 18 per medium and large farm) were collected. A questionnaire was administered at

J. Food Prot., Vol. 77, No. 9

each farm to obtain demographic data and information about general operations. (ii) At the port of entry (airports), imported eggs (table and hatching), samples of fecal matter, and samples of bedding papers in boxes used to import day-old chicks were collected. (iii) At the hatchery, fertile hatching eggs (preincubation), meconium samples from day-old chicks, broken egg shells, and environmental swabs from hatchers and incubators were collected. Of the six layer hatcheries in the three countries (two in Trinidad and Tobago, one in Grenada, and three in St. Lucia), only one hatchery in St. Lucia was in operation (i.e., hatching eggs) during the scheduled sampling periods. A total of 38 samples (6 pooled hatching egg contents, 6 pooled egg shells, 20 meconium, and 6 environmental) were collected. (iv) In hotel kitchens, eggs stored in refrigerators and environmental swabs of all refrigerators and freezers were collected during the visits. Collection of samples: layer farms. To collect cloacal samples, sterile swabs were inserted into the cloacae of a randomly selected number of birds. The swabs were rotated inside the cloaca, placed into transport medium, and held in a cooler for transportation to the laboratory. To collect environmental samples (drag swabs), a sterile surgical gauze swab (10 by 10 cm) attached to 110 cm of string and stapled to a wooden tongue depressor was moistened in sterile buffered peptone water (BPW) and then dragged along the sides and through the middle of the pen. The swab was then placed in 20 ml of BPW as transport medium and held in a cooler with ice packs for transportation to the laboratory. Table eggs selected randomly from stacks of crates were removed with sterile gloves and placed in sterile containers. The containers were then wrapped in sterile aluminum foil and placed in a cooler for transportation to the laboratory. Approximately 50 g each of different types of feed used on the farm was aseptically collected and placed into sterile plastic containers. Collection of samples: port of entry. Twenty boxes (or as many boxes available if there were fewer than 20) of day-old chicks were selected randomly at the airport. From each box, two swabs of fecal material were collected and placed in commercially available transport medium (Copan, Brescia, Italy). Both stained and unstained shredded bedding paper from each box was removed and placed in a sterile fecal cup and appropriately labeled. Twelve eggs selected randomly were removed with sterile gloves, placed in sterile containers, and held in a cooler for transportation to the laboratory. Collection of samples: hotel kitchens. Twelve table eggs selected randomly from stack of crates were removed with sterile gloves and placed into sterile containers. The containers were then wrapped in sterile aluminum foil and placed in a cooler for transportation to the laboratory. To collect environmental samples, sterile cotton-tipped applicators were moistened in sterile saline and rotated along the entire area of the handles of all refrigerators, using a new swab for each refrigerator. Each swab was placed in 20 ml of sterile saline and transported to the laboratory on ice. A similar procedure was used for freezers. Collection of samples: hatcheries. To collect environmental samples, sterile cotton-tipped applicators were moistened in sterile saline and used to swab the entire interior of each hatcher. The


swabs taken for each hatcher were then placed in 20 ml of sterile saline and transported to the laboratory on ice. Six eggs selected randomly were removed with sterile gloves and placed into sterile containers. The containers were then wrapped in sterile aluminum foil and placed in a cooler for transportation to the laboratory. To collect meconium samples from hatched birds, a sterile swab was rotated on the meconium samples, placed into transport medium, and transported to the laboratory on ice. Five samples of broken egg shells from the hatcher trays were aseptically collected and compressed into sterile fecal cups for transportation to the laboratory. Processing of samples. Samples preenriched in BPW (eggs, environmental drag samples, feeds, broken egg shells, and environmental swabs from hotels and hatchers) were selectively enriched in tetrathionate (TT) broth (Oxoid, Basingstoke, UK) and Rappaport-Vassiliadis (RV) broth (Oxoid) and incubated for 18 to 24 h at 37 and 42uC, respectively. Processing of samples: shell eggs. Six eggs were pooled for each sample. Moistened sterile swabs were applied to the entire external surface of each egg and subsequently dipped in 20 ml of sterile saline as previously described (2). The eggs were then disinfected by submerging them in 70% alcohol for 10 min and then air dried. Each egg was then flamed at the pointed end and cracked with a sterile scalpel blade, and the contents were poured into a stomacher bag. The pooled egg contents were homogenized for 60 s at low speed in a Seward 400 Lab Stomacher (Seward, London, UK). Twenty-five milliliters of pooled egg contents was then mixed with 225 ml of BPW and incubated at 35uC for 18 h. Ten milliliters of the pooled egg shell swab rinsate was placed in 90 ml of BPW and incubated for 35uC for 18 h. Processing of samples: environmental drag samples. The drag swab sample from each pen was placed in 250 ml of BPW and incubated at 35uC for 18 h for enrichment. Processing of samples: cloacal, meconium, and fecal swabs. Each swab was mixed with 10 ml of sterile saline, and 0.5 ml of this suspension was then added to 9.5 ml of TT broth and 9.5 ml of RV broth and incubated for 24 h at 35 and 42uC, respectively. Processing of samples: chick bedding paper. Approximately 5 g of the shredded chick bedding paper with stained and unstained areas was added to 15 ml of sterile saline and mixed with a vortex mixer, and 0.5 ml of this suspension was added to 9.5 ml of TT broth and 9.5 ml of RV broth and incubated as described. Processing of samples: feed and broken egg shells. Each 25-g sample of feed and egg shells was aseptically weighed, placed in a stomacher bag containing 225 ml of BPW, and homogenized for 2 min. The homogenized mixture was then placed into a sterile flask and incubated at 35uC for 18 h. Processing of samples: environmental swabs (hotel kitchens and hatcheries). Ten milliliters of suspension from each swab was added to 90 ml of BPW and incubated at 35uC for 18 h. Isolation, identification, and serotyping of Salmonella. All selective enrichment broths were subcultured onto xylose-lysinedesoxycholate agar (XLD; Oxoid) and brilliant green agar (BGA; Oxoid) and incubated aerobically at 37uC for 24 h. Suspect colonies of Salmonella on XLD and BGA were subjected to standard biochemical tests (4, 24). Isolates biochemically identified

SALMONELLA IN LAYER FARMS IN THE CARIBBEAN

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as Salmonella were subjected to a slide agglutination test using a commercial kit with Salmonella polyvalent antiserum (A-I & Vi; Difco, BD, Detroit, MI). All slide-positive isolates were confirmed as Salmonella and serotyped at the Caribbean Epidemiology Center (Port of Spain, Trinidad and Tobago). Statistical analyses. Data were analyzed with the Statistical Package for Social Sciences (SPSS), version 22.0 (IBM, Armonk, NY). Logistic regression analysis was performed to determine which independent variables, i.e., country of origin, type of farms (large, medium and small), type of samples (cloacal swabs, environmental swabs, feed, eggs, hatchery, imported day-old chicks), and risk factors for Salmonella were predictive for the presence of Salmonella, the dependent variable. A forward stepwise method was used to test variables for entry into the model (at P , 0.05), and independent variables were removed (P . 0.10) based on the significance of the change in the likelihood ratio. Because explanatory variables are often correlated, the stepwise method was used because it combines both the forward selection and backward elimination.

RESULTS Demographic data and management and production practices in layer farms in three Caribbean countries. The demographic data and management and production practices for the three countries are shown in Table 1. Layer farms in Trinidad and Tobago were significantly larger (P ~ 0.000); 6 (26.1%) of 23 farms in this country had over 20,000 birds, whereas none of the 21 and 37 layer farms in Grenada and St. Lucia, respectively, were that large. The frequencies of four practices (housing system, age pullets start to lay, age when layers are culled, and preparation of eggs on farms) were significant within each of the three countries (P , 0.05) but not across countries (P . 0.05), but the frequencies of two practices (preparation of eggs on farms and storage of feeds) were significantly different (P , 0.05) both within and across countries. Practices used to prevent disease on farms. The frequency of disease preventive measures is displayed in Table 2. Of the 10 practices investigated, the frequencies of 4 (vaccine administered, sanitation procedure, types of pest control, and biosecurity measures) were significantly different (P , 0.05) within and across the three countries. Risk factors for Salmonella infection in layers, feed, and farm environments. Table 3 shows the frequency of isolation of Salmonella based on pooled data from layers, feeds, and farm environments in the three countries. Of the eight factors studied, farm size was the only one that significantly affected (P ~ 0.031) the frequency of isolation of Salmonella. Isolation of Salmonella from layer farms. In Trinidad and Tobago, the frequency of detection of Salmonella on poultry farms and their environments was significantly higher (P ~ 0.024) on large farms (100.0%) than on medium farms (0.0%) and small farms (33.3%); however, these differences were not significant in Grenada (P ~ 0.400) and St. Lucia (P ~ 0.275) (Table 4). The overall frequency of isolation of Salmonella was 40.0% (14 of 35

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TABLE 1. Demographic data and management and production practices on layer farms in three countries Trinidad and Tobago Parameter

No. of farms No. of layers on farm ,1,000 1,001–5,000 5,001–10,000 10,000–20,000 .20,000 No response Breed of layer hens used Hyline Brown Bovan Brown ISA Brown Mixed Other or no response Other animals on farma Yes No Housing system Cages Pens, deep litter Mixture Age groups present on farm One age group Several age groups No response Supplier of chicks Local Imported from the United States Imported from the Caribbean region Local and imported from the Caribbean region Imported from both the Caribbean region and the United States Others or no response Age pullets start to lay (wk) 16–17 18–24 25–30 No response Age at culling of layers (mo) 12–13 14–17 18–24 .25 or no response Preparation of eggs on farm Yes No No response Methods used to prepare eggs for saleb Dry clean by hand Wiping with damp cloth Machine wash with warm water Washed with water Wipe with bleach Wash with water and hand sanitizer Other or no responsec Destination of eggs from farmd Supermarkets Community

No (%)

P value

23

Grenada No (%)

21 (8.7) (26.1) (17.4) (17.4) (26.1) (4.3)

0.012 12 (57.1) 8 (38.1) 1 (4.8) 0 0 0

0.043 4 3 5 10 1

(17.4) (13.0) (21.7) (43.5) (4.3)

(33.3) (19.0)

15 0 16 3 3

(38.1) (9.5)

0.144

0.000

0.345

0.732

0.004

0.441

0.000

0.000

0.057

0.000

0.412

0.000

0.191

0.000

0.000

0.040

0.000

(8.1) (81.1) (8.1) (2.7)

0.000 0 3 (8.1) 30 (81.1) 4 (10.8) 0.001 18 (85.7) 3 (14.3) 0

0.000

33 (89.2) 1 (2.7) 3 (8.1) 0.000

0 20 (95.2) 0 0 1 (4.8) 0 0 0.022

21 (91.3) 3 (13.0)

3 30 3 1

(4.8) (4.8) (81.0) (9.5)

0.000

(47.6) (14.3) (4.8) (9.5) (4.8) (4.8) (14.3)

0.000

6 (16.2) 9 (24.3) 0.000

0.000

21 (91.3) 2 (8.7) 0

0.187

15 (40.5) 4 (10.8) 3 (8.1) 0

1 (4.8) 20 (95.2) 0 0 1 1 17 2

0.869

(43.2) (8.1) (8.1)

0.009 (14.3) (4.8) (42.9) (28.6)

0.022

0 1 (4.3) 22 (95.7) 0

0.000

16 (43.2) 18 (48.6) 3 (8.1)

1 (4.8) 1 (4.8)

6 (26.1) 17 (73.9) 0 0

0.001 (40.5)

0.000

0.144

0 0

0.000

0 34 (91.9) 3 (8.1)

2 (9.5) 19 (90.5) 0 3 1 9 6

0.001

(2.7)

0.000

0.002

8 (34.8) 15 (65.2) 0 0

All countries (P value)

18 (48.6) 19 (51.4)

0 21 (100.0) 0

4 (17.4) 19 (82.6) 0

P value

(35.1) (45.9) (10.8)

0.050 15 (71.4) 6 (28.6)

1 (4.3) 20 (87.0) 2 (8.7)

10 3 1 2 1 1 3

13 17 4 0 0 1 0.228

7 4 0 8 2

15 (65.2) 8 (34.8)

No (%)

37

0.637 2 6 4 4 6 1

P value

St. Lucia

21 5 0 0 0 1 10

(58.3) (13.9)

(2.8) (27.8)

0.275 8 (38.1) 12 (57.1)

11 (29.7) 18 (48.6)



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TABLE 1. Continued Trinidad and Tobago Parameter

Hotel Other or no response Supplier of feeds Local Imported Mixture of local and imported Storage of feeds Store room or shed Other Pen No response

No (%)

P value

0 4 (17.4)

Grenada No (%)

P value

2 (9.5) 6 (28.6) NAe

23 (100.0) 0 0

No (%)

NA


0.000

0.032

0.000

0.010

29 (78.4) 7 (18.9) 1 (2.7) 0.000

17 (81.0) 1 (4.8) 3 (14.3) 0

P value

11 (29.7) 8 (21.6)

21 (100.0) 0 0 0.000

16 (69.7) 6 (26.1) 0 1 (4.3)

St. Lucia

34 (91.9) 1 (2.7) 2 (5.4) 0

a

Trinidad and Tobago: pigs (13.3%), dogs (86.7%), and cats (40.0%). Grenada: pigs (14.3%), dogs (47.6%), cats (19.0%), cows (14.3%), sheep (9.5%), and goats (28.6%). St. Lucia: pigs (22.2%), dogs (66.7%), cats (27.8%), cows (5.6%), sheep (22.2%), and goats (27.8%). b For Trinidad and Tobago, only 21 farms were asked for responses to this question. c Trinidad and Tobago: wash, oil, and crate (4.8%); dry clean, oil, and grade (9.6%). St. Lucia: wash, oil, and crate (2.8%); clean, pack, and distribute (13.9%); preclean with steel wool and wash in egg washer (2.8%); no response (8.3%). d Eggs from some farms had multiple destinations. e NA, not applicable because the variable was constant and the chi-square test could not be performed.

farms) for the three countries, with a range of 26.1% (small farms) to 77.8% (large farms), and this difference was significant (P ~ 0.025). Frequency of isolation of Salmonella by source on layer farms. Within each of the three countries, the frequency of isolation of Salmonella was higher in environmental samples than in cloacal swabs and feeds, but the differences were significant only in Trinidad and Tobago (P ~ 0.000) and St. Lucia (P ~ 0.000) (Table 5). Although the frequency of isolation of Salmonella was highest for cloacal swabs in St. Lucia (1.6%), for environmental swabs in Trinidad and Tobago (26.7%), and for feeds in Trinidad and Tobago (6.5%), the differences for each type of sample across countries were not significant (P . 0.05). Pooled samples of freshly laid eggs (6 eggs per pooled sample) were positive for Salmonella only in Trinidad and Tobago, with a frequency of 12.5% (3 of 24 pooled samples [18 of 144 eggs]), and all Salmonella isolates were recovered from egg shell surfaces (Table 6). All 12 pooled samples (72 eggs) from Grenada and 23 pooled samples (138 eggs) from St. Lucia were negative for Salmonella. Frequency of isolation of Salmonella from samples collected at ports of entry (airports). All 200 samples collected from the airports (fecal swabs of chicks, bedding paper in shipment boxes, and eggs) were negative for Salmonella. Frequency of isolation of Salmonella from a hatchery in St. Lucia. All 38 samples, i.e., 6 pooled egg samples, 6 egg shell samples, 20 meconium samples, and 6 environmental samples, were negative for Salmonella. Frequency of isolation of Salmonella from table eggs and kitchen environments of hotels. All samples collected

from hotel kitchens in all three countries were negative for Salmonella. Serotypes of Salmonella isolates recovered from various sources. For cloacal samples from layers, the Salmonella serotypes recovered were Anatum 3,10:e,h:1,6 in Trinidad and Tobago, group C in Grenada, and Kentucky (8),20:i:z6 in St. Lucia (Table 6). Three Salmonella serotypes (Mbandaka 6,7:z10:e,n,z15, Polyvalent A–negative, and Montevideo 6,7:g,m,s:) were isolated from freshly laid eggs on layer farms only in Trinidad and Tobago. Eight Salmonella serotypes were isolated from environmental swabs in Trinidad and Tobago, and Anatum 3,10:e,h:1,6 was dominant. In St. Lucia, the dominant Salmonella serotype was Kentucky (8),20:i:z6. From all sources in all the three countries, 35 Salmonella isolates were recovered, only one of which (2.9%) was identified as Salmonella Enteritidis 1,9,12:g,m:, which represented only 6.6% of the total 15 serotypes isolated. DISCUSSION The demographic data revealed that the layer poultry industry in these three countries were similar but with some differences that could affect the exposure of farms to Salmonella. For example, farms in Trinidad and Tobago were considerably larger than those in the other two countries, and the isolation rate for Salmonella was significantly higher on large farms (77.7%) than on medium and small farms (26.9%). This finding is in agreement with published reports in which poultry farm size was reported as a risk factor for Salmonella infection (12, 36). In Trinidad and Tobago, a majority of the farms imported day-old chicks from the United States, whereas farmers in Grenada and St. Lucia imported chicks primarily

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TABLE 2. Farm practices used to prevent disease in three countries Trinidad and Tobago Practice

No. of farms Vaccine administereda Yes No Do not know or no response Location of pullets Transferred to a laying unit Stay in same pen, cage, area Other or no response Sanitization procedures Change litter Change litter and wash Change litter and clean with disinfectant or bleach Other or no response Water supply Government pipe Rain Water system Automatic Manual Automatic and manual Other or no response Presence of pests Yes No No response Type of pests presentc Rats Birds Mice Other Type of pest control Poison Bait Other Biosecurity measures Foot dips Restricted access Protective clothing Other or no response Disease outbreak in last 10 yr Yes No a b c

No. (%)

P value

23

Grenada No. (%)

St. Lucia P value

21 0.000 1 (4.8) 20 (95.2) 0 0.007

5 (21.7) 18 (78.3) 0

0.000

0.000

0.000 1 (4.8) 0

2 (5.4) 25 (67.6)

16 (69.6) 6 (26.1)

19 (90.5) 1 (4.8)

6 (16.2) 4 (10.8)

NAb

0.000 20 (95.2) 2 (9.5)

0.003

NA

0.000

(90.0) (60.0) (15.0) (15.0)

0.000 1 (5.0) 17 (85.0) 3 (15.0)

14 6 7 3

0.003

0.000

0.000

0.356

0.000

0.000

0.732

0.000

0.059

0.130

0.003

0.000

0.001

0.000

0.001

0.580

5 (27.8) 3 (16.7) 9 (50.0) 0.000

0 20 (95.2) 1 (4.8) 0 0.000

2 (8.7) 21 (91.3)

0.000

(77.8) (33.3) (38.9) (16.7)

0.005 19 (95.0) 2 (10.0) 3 (15.0)

(34.8) (78.3) (4.3) (4.3)

0.132

18 (48.6) 16 (43.2) 3 (8.1) 0.000

18 12 3 3

0.000

(5.4) (83.8) (2.7) (8.1)

0.000

0.002

8 18 1 1

2 31 1 3

20 (95.2) 1 (4.8) 0

18 (90.0) 8 (40.0) 0 2 (10.0)

0.000

36 (97.3) 3 (8.1)

21 (100.0) 0 0 0

20 (87.0) 3 (13.0) 0

0.000

3 (8.1) 33 (89.2) 1 (2.7)

1 (4.3) 0

15 (65.2) 5 (21.7) 0 3 (13.0)


1 (2.7) 35 (94.6) 1 (2.7)

7 (33.3) 13 (61.9) 1 (4.80

23 (100.0) 0

P value

37

0.000 20 (87.0) 2 (8.7) 1 (4.3)

No. (%)

4 5 21 8

(10.8) (13.5) (56.8) (21.6)

0.000 1 (4.8) 20 (95.2)

1 (2.7) 36 (97.3)

Vaccination against Marek’s disease, Newcastle disease, pox, bursal disease, bronchitis, and egg drop syndrome. NA, not applicable because the variable was constant and the chi-square test could not be performed. For Trinidad and Tobago, Grenada, and St. Lucia, only 20, 20, and 18 farms, respectively, responded to this question.

from regional suppliers. These differences in sources of dayold chicks can affect the introduction of Salmonella into layer farms in these three countries. However, all day-old chicks and table eggs imported from the United States and regional sources were negative for Salmonella. Imported day-old chicks have been responsible for the introduction of Salmonella into other countries, with carriage rates of 12.6 to 19.2% (6, 31).

The overall frequency of isolation of Salmonella from the cloacae of laying birds was relatively low (0.9%) in these three countries, which was considerably lower than the range (4 to 12%) reported for other countries (3, 16). Swabs of litter in pens on layer farms in Trinidad and Tobago had significantly higher frequency (26.7%) of isolation of Salmonella than did those in Grenada (6.5%) and St. Lucia (16.1%), which is an indication that freshly



TABLE 3. Risk factors for Salmonella on all farms in Trinidad and Tobago, Grenada, and St. Lucia

Risk factor

Farm sizea Large Medium Small Age groups of layers on farms One age group only Several age groups Source of day old chicks United States Caribbean region Other Vaccinationb Yes No Sanitization procedures Change litter Change litter and wash Change litter and clean with disinfectant, bleach Other or no responsec Presence of pests Yes No Biosecurity measures Restricted access Protective clothing (aprons, boots, gloves) Multiple (various combinations of the above) None Other Disease outbreaks in last 10 yr Yes No

No. of farms tested

No. (%) of farms positive for Salmonella

9 3 23

7 (77.8) 1 (33.3) 6 (26.1) 0.627

5 30

1 (20.0) 13 (43.3)

15 14 6

7 (46.7) 3 (21.4) 4 (66.7)

0.156

0.206 11 24

6 (54.5) 8 (33.3)

2 4

0 2 (50.0)

14 15

3 (18.8) 9 (60.0)

0.119

0.321 30 5

13 (43.3) 1 (20.0)

17

7 (41.2)

10 4 2

0

2 (20.0) 3 (75.0) 2 (100.0) 0.348

3 32

Farm size

Trinidad and Tobago Large Medium Small Total Grenada Large Medium Small Total St. Lucia Large Medium Small Total All countries Large Medium Small Total a

0.069

2

TABLE 4. Frequency of isolation of Salmonella from layer farms in three countries

P value

0.031

2 (66.7) 12 (37.5)

a

Large, .10,000 birds; medium, 5,001 to 10,000 birds; small, ,5,000 birds. b Vaccination against Marek’s disease, Newcastle disease, pox, bursal disease, bronchitis, and egg drop syndrome. c Includes a combination of practices.

laid eggs in Trinidad and Tobago have a higher risk of being contaminated by Salmonella. This risk was confirmed by the finding that in only Trinidad and Tobago were the shells of freshly laid eggs contaminated with Salmonella. Wood shavings and sand used as litter on layer farms may be contaminated by pests such as rodents (22, 23, 38) and could have occurred in Trinidad and Tobago, where 90% of the farms reported a rat problem. The need for good biosecurity practices and disinfection cannot be overemphasized, particularly between introducing new batches of birds to the pens to avoid residual contamination from the preceding flocks (17). The overall frequency of isolation of

1477

Total no. of farms

No. (%) of farms sampled

No. (%) of farms positive for Salmonellaa

P value

23

5 2 3 10 (43.5)

5 (100.0) 0 1 (33.3) 6 (60.0)

0.024

21

0 1 9 10 (47.6)

NA 1 (100.0) 3 (33.3) 4 (40.0)

0.400

37

4 0 11 15 (40.5)

2 (50.0) NA 2 (18.2) 4 (26.7)

0.275

81

9 3 23 35 (43.2)

7 1 6 14

(77.8) (33.3) (26.1) (40.0)

0.025

Based on results from cloacal swabs, eggs, feeds, and environmental samples from the layer farms. NA, not applicable.

Salmonella from farm environments in the current study (17.8%) is within the range of 0.95 to 33.3% reported for other countries (6, 34). The finding that only the pooled egg shells (12.5%) and not the pooled egg contents yielded Salmonella in Trinidad and Tobago suggests horizontal rather than transovarian transfer. This finding differs from those of two previous studies conducted in Trinidad and Tobago. Indar et al. (20) isolated Salmonella from 4.7% of egg shell samples and 1.2% of egg content samples, and Adesiyun et al. (2) reported Salmonella isolation rates of 6.5% for both pooled egg shells and egg contents. Because the same isolation techniques were used in all three studies, the differences found should not have been due to the isolation protocols, although differences in techniques can affect the recovery rates and serotypes of Salmonella (5). Therefore, the increase in horizontal transmission of Salmonella in in Trinidad and Tobago appears to be real. Reports elsewhere have documented contamination rates of eggshells at 0 to 34% (3, 6, 28). All 31 pooled samples of eggs (shells and egg content) from 19 hotel kitchens in the three countries were negative for Salmonella, which is important for food safety. Although 12.5% of the freshly laid eggs sampled on farms in Trinidad and Tobago were positive for Salmonella, all table eggs sampled from hotel kitchens in that country were negative for the pathogen. This difference could be in part due to the practice of preparing eggs (cleaning and sometimes disinfecting) for sale to hotels, supermarkets, and other destinations. Another possibility is that the farms

0.000

0.091 0.074 0.333 0.494 7 (1.6) 5 (16.1) 0 12 (2.5) 0.111

437 31 20 488 2 (0.9) 2 (6.5) 0 4 (1.5) 227 31 13 271 0.000

P value No. of samples tested

454 45 31 530

Sample type

Cloacal swabs of layers Environmental swabs Feed Total

No. (%) samples positive for Salmonella

(0.2) (26.7) (6.5) (2.8)

P value No. (%) samples positive for Salmonella No. of samples tested

Grenada Trinidad and Tobago


1 12 2 15

No. (%) samples positive for Salmonella No. of samples tested

St. Lucia

P value


ADESIYUN ET AL.

TABLE 5. Frequency of isolation of Salmonella by source on layer farms in three countries

1478

with Salmonella-positive eggs did not supply eggs to the hotels that provided eggs for this study. The low prevalence (12.5%) may reflect a reduction of contamination of eggs on poultry farms in Trinidad and Tobago, this hypothesis must be confirmed with further work because the present prevalence study provided information on the contamination of eggs at only a single time point. Salmonellosis associated with contaminated eggs and egg products has been reported in the Caribbean region (8, 21) and elsewhere (11, 19, 26, 30, 32). However, sample size, isolation techniques used, and time frame should be considered when comparing prevalence data from various studies. Feeds, particularly as part of a deep litter management system, can be a source of contamination of eggs laid in the litter; 6.5% of feed samples from Trinidad and Tobago were positive for Salmonella. Other researchers have recovered Salmonella from feeds provided for layers on farms at frequencies of 0.1 to 5.6% (6, 17, 34), indicating that feed can serve as a vehicle for Salmonella transmission on poultry farms. The recovery of distinctly different Salmonella serotypes from various poultry sources in these three countries has epidemiological relevance and may indicate the origins and spreading mechanisms of Salmonella serotypes to the layer farms. Eggborne human salmonellosis in these countries is most likely caused by these country-prevalent serotypes. The relatedness of the Salmonella isolates recovered from cloacae and environmental sources cannot be conclusively established because genetic fingerprinting was not performed. Adesiyun et al. (1) used pulse-field gel electrophoresis to determine the relationships among Salmonella Enteritidis isolates recovered from each of the Caribbean countries studied. Salmonella serotypes differ among layer farms and countries (9, 28, 29, 34), and all serotypes recovered in the present study have been documented previously in the Caribbean region (1, 10, 20). Only 2.9% (1 of 35) of all isolates were Salmonella Enteritidis, representing 6.7% of the 15 serotypes recovered in the current study. This finding is not in accordance with published reports from Trinidad and Tobago, in which this the serotype accounted for 58.3% of all the Salmonella isolates recovered from table eggs in this country (2), nor with reports from outside the region (25, 33, 39). This difference may reflect a change in production and management practices, resulting in a change in the epidemiology of this egg- and layer-associated serotype, which needs further investigation. However, the difference also might be an artifact of the limited time period over which the study was conducted and the small number of samples that were positive for Salmonella. This survey revealed that the poultry farm environments in the three countries studied pose a risk for contaminating freshly laid eggs, although table eggs (local and imported) and imported day-old chicks appear to pose minimal risk for Salmonella transmission through consumption of eggs or the introduction of the pathogen into the respective countries. The very low frequency of detection of Salmonella Enteritidis from all sources indicates that this serotype may not be commonly associated with eggborne salmonellosis in the Caribbean region. These results revealed the presence of country-specific Salmonella serotypes associated with

b

Anatum 3,10:e,h:1,6 Mbandaka 6,7:z10:e,n,z15 Polyvalent A–negative Montevideo 6,7:g,m,s: Groups I–O (1) Enteritidis 1,9,12:g,m: Anatum 3,10:e,h:1,6 Group E Caracas 6,14.(25):g,m,s Braenderup Uganda 3,10:l,z13:1,5 Muenster 3,10:e,h:1,5 Anatum 3,10:e,h:1,6 Uganda 3,10:l,z13:1,5

Salmonella serotypes

1 1 1 1 1 1 4 1 2 1 2 1 1 1

No. of isolates

4

0

2

2 0

No. (%) of samples positive for Salmonella

NA

Group C Group D

Group C NA


Grenada

1 1

2

No. of isolates

12

0

5

7 0


NA

Kentucky (8),20:i:z6 Group B

Kentucky (8),20:i:z6 NA


St. Lucia

3 2

7

No. of isolates

Salmonella isolates were recovered from different farms. Pooled samples of freshly laid eggs (6 eggs per pooled sample) were tested for Salmonella. In Trinidad and Tobago, 3 of 24 pooled samples (18 of 144 eggs) were positive (frequency of 12.5%), and all Salmonella isolates were recovered from egg shell surfaces. None of the 12 pooled samples (72 eggs) from Grenada and none of the 23 pooled samples (138 eggs) from St. Lucia were positive for Salmonella.

19

2

Feed

Total

13

Environmental swabs

a

1 3

Cloacal swabs of layers Eggs on the farmb

Sample type


Trinidad and Tobago

TABLE 6. Serotypes of Salmonella isolated from various sources in three countries a

J. Food Prot., Vol. 77, No. 9 SALMONELLA IN LAYER FARMS IN THE CARIBBEAN

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1480

ADESIYUN ET AL.

poultry farms in these three countries, and these serotypes may play a role in salmonellosis in humans and animals. ACKNOWLEDGMENTS The authors are grateful to the Caribbean Food Research Institute (CFRI), Jamaica, and the Pan American Health Organization (PAHO) (grant 65503 52163) for funding the project. The assistance provided by Ms. Margaret Kalloo (Caribbean Community Secretariat) by circulating the questionnaire to the regional chief veterinary officers is appreciated. We thank the CVOs of Trinidad and Tobago, Grenada, and St. Lucia, who assisted in sample collection.

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