Occurrence of Salmonella sp in Laying Hens

This study was carried out to investigate the presence of Salmonella sp in flocks of white laying hens. In different farms, the transport boxes of twelve flocks were inspected at arrival for the presence of Salmonella. Four positive (A, B, L and M) and one negative (I) flocks were monitored at each four weeks using bacteriological examination of cecal fresh feces up to 52 weeks. Birds were also evaluated at 52 weeks, when 500 eggs were taken randomly, and at 76 weeks, after forced molt. Salmonella enterica serovar Enteritidis and S. enterica rough strain were isolated from the transport boxes of the four positive flocks (flocks A, B, L and M). Salmonella sp was not isolated from the transport boxes or from the feces after 76 weeks-old in flock I. Salmonella sp was isolated in the 1, 11, 34, 42 and 76 weeks from flock A; in the 1, 4, 11 and 76 weeks from flock B; in the first week and in the 17 to 52 weeks from flock L; and in the 1 and 76 weeks from flock M. S. Enteritidis, S. enterica rough strain and Salmonella enterica serovar Infantis were isolated from the four positive flocks. Besides, Salmonella enterica serovar Javiana was isolated from flocks B and L, and Salmonella enterica serovar Mbandaka was isolated from flock L. Eggs produced by flock A and by flock L were contaminated with S. Enteritidis and S. enterica rough strain. According to these results, Salmonella-infected flocks may produce contaminated eggs.


INTRODUCTION
Salmonella remains the main food borne bacterial diseases in human (Barrow, 2000), and many of the world outbreaks are related to food containing poultry products (Rodrigue et al., 1990;Cox, 1995).Besides, infected birds may develop the illness (Suzuki, 1994).Fowl are the specific host of Salmonella enterica serovars Pullorum and Gallinarum, which cause pullorum disease and fowl typhoid, respectively.Other serotypes with no specific host, such as Enteritidis and Typhimurium, may infect chickens and persist in the final poultry product, inducing or not clinical disease during rearing.Thus, the control of Salmonella in poultry flocks is crucial for poultry industry success.
Salmonella are introduced in poultry farms by several ways, including day-old infected chicks, domestic animals, human, equipment, water and feed (Barrow, 2000).Once the farm is contaminated, it is very difficult to eliminate Salmonella from the environment (Davies & Wray, 1995b).Factors such as the presence of wild birds, rodents, domestic animals and insects, as well as intensive production systems and multiple age flocks keep Salmonella on farms, which compromise eradication methods (Berchieri Junior & Barrow, 1995).
Paratyphoid salmonellas are responsible for poor performance of breeders, decreased egg production by layers, infertility and mortality.Furthermore, there is restriction for commercialization and egg consumption also decreases (Barrow, 2000).Salmonella infections may cause systemic disease in young and adult chickens, associated with clinical symptoms and lesions in organs like liver, spleen, heart, and mostly the ceca (Suzuki, 1994;Barrow, 2000).In addition, with or without clinical disease, Salmonella shedding occurs and the laid egg becomes contaminated (Suzuki, 1994;Barrow, 2000).
Infected breeder flocks are responsible for vertical transmission.The eggs are contaminated either from the ovary tissue or on their passage through the cloaca (Nakamura et al., 1993).Vertical transmission of Salmonella was reported by Zancan et al. (2000), who found 44.45 % of positive transport boxes, and horizontal transmission of Salmonella enterica serovar Enteritidis (SE) was demonstrated by Gast & Holt (1999) and Soncini et al. (2000).They reported that birds free of bacteria in contact with inoculated birds became infected and excreted SE in the feces within the following 12 to 24 hours.The duration of fecal excretion is not easily defined.According to Gast & Holt (1998), laying hens experimentally exposed to SE soon after birth may remain infected until maturity, producing contaminated eggs and eliminating the bacteria to the environment.Conversely, Berchieri Júnior et al. (2001) reported that chicks orally infected with SE at 7 days of age presented systemic infection, and the bacteria were isolated from liver and ceca for 10 weeks, but not after this period.
Since the 80´s, the number of human food borne salmonellosis has increased worldwide and has been mostly related to the consumption of poultry products (Rodrigue et al., 1990;Taunay et al., 1996).On the other hand, it was demonstrated that the food was prepared with raw egg contaminated by SE (Duguid & North, 1991;Mishu et al., 1991;Cox, 1995;Barrow, 2000).
All control programs of infectious diseases are based on the knowledge of the routes of introduction and pathogen dissemination.In view of the lack of information about the epidemiology of avian salmonellosis in commercial poultry in Brazil, this work was carried out to investigate the presence of Salmonella in naturally infected flocks of commercial laying hens from newly hatched chicks up to 76 weeks of age.The presence of Salmonella was also investigated in eggs laid by hens at 56 weeks of age.

MATERIAL AND METHODS
Twelve flocks of white laying hens were inspected prior the beginning of the experiment (Table 1).Salmonella was searched inside all transport boxes when one-day-old chicks arrived at different farms (100 chicks/ box).Four Salmonella-positive flocks and one negative flock were used in the experiment.Samples of feces were collected for bacteriological examination starting on the seventh day and at every four weeks thereafter, up to 52 weeks.Additional evaluations were performed in 500 eggs laid by hens with 52 weeks of age, and after molting in fecal samples from 76-week-old birds.
Experimental procedure was done according Wray and Davies (1994) recommendations.

Sampling of transport boxes
Samples were taken from the internal wall and bottom of the transport boxes using one large gauze swab per box, moistened in PBS pH 7.4.Five swabs were placed in a glass jar containing 100mL selenite broth (CM395) plus novobiocin (40mg/L) and incubated overnight at 42ºC.The broth was plated on brilliant green agar (Oxoid CM263) and MacConkey agar (Oxoid CM115) and plates were incubated overnight at 42ºC.Suspected colonies were inoculated in TSI agar (Oxoid CM277) and lysine iron agar (CM281), incubated overnight at 37ºC and confirmed using polyvalent sera against O and H Salmonella antigens (Probac).Complete identification and serotyping of the isolates were performed at the Adolfo Lutz Institute in São Paulo, Brazil.

Feces
Fresh samples of cecal feces were collected under the cages of the birds.Each sample corresponded to 200 birds up to 17 weeks and 100 birds afterwards.The samples were processed according to the procedure described above.

Eggs
Eggs were collected and placed in sterile trays, The trays were put in plastic bags and taken to the laboratory under refrigeration.Each egg was placed in a sterile glass jar and broken by agitation.The contents were mixed and the jars were incubated at 37ºC for 24 h.Bacteriological procedure was the same described above, except that Hektoen agar (Oxoid CM409) was used instead MacConkey agar.

RESULTS
The presence of Salmonella was investigated in 614 boxes corresponding to 12 flocks of one-day-old birds.Salmonella Enteritidis was detected in four flocks (129 boxes).In one of these flocks, S. enterica rough strain (R strain) was also isolated (Table 1).Samples were collected from four positive flocks (A, B, L and M) and one negative flock (I) until the end of the experiment (Table 2).The positive flocks showed the same result at the second sampling, but isolation varied afterwards.In flock A, Salmonella was isolated from fecal samples at 11, 34, 42 and 76 weeks of age.Salmonella was isolated from fecal samples collected from flock B at 4, 11 and 76 weeks of age.Flock L presented positive results for Salmonella isolation in all samplings from 17 to 52 weeks of age.Flock L was not evaluated at 76 weeks of age because it was depopulated.Salmonella was not isolated from flock M between 4 and 52 weeks of age, but it was detected at 76 weeks of age.Flock I presented negative results throughout the experimental period, even after molting.Several serovars of Salmonella were isolated (Table 3).S. Enteritidis, rough strain and S. enterica serovar Infantis were isolated from fecal samples from the four positive flocks, S. enterica serovar Javiana was isolated from fecal samples from flocks B and L, and S. enterica serovar Mbandaka was isolated only from flock L.
Salmonella was studied in 500 eggs from each flock (Table 4).SE and R strain were found in one egg from flock A (0.2%) and R strain was also isolated from 10 eggs from flock L (2.0%).

DISCUSSION
Salmonella was detected in four transport boxes (33.3%) from a total of 614 boxes with one-day-old laying hens.Similarly, Salmonella was found in 20% of the samples of meconium and fluff (Bhatia & Mcnabb, 1980), 17% of the samples taken from commercial hatchery residues (Bailey et al., 1994), 26% of the samples of transport boxes and egg shell (Cox et al., 1997) and 44.45% of transport boxes (Zancan et al., 2000).The presence of Salmonella sp in transport boxes of day-old birds strongly suggests the occurrence of vertical transmission (Lister, 1988;Mc Ilroy & Mac Craken, 1990).Bacteria are present in chicks for a long period of time when they are exposed to Salmonella at the end of the hatchery period or during the first hours of life, and may be disseminated to other susceptible chicks in the same flock or other flocks (Gast & Holt, 1998).Therefore, the first step to prevent Salmonella introduction in farms is to obtain Salmonellafree chicks, avoiding lateral transmission.Since there are many sources of Salmonella contamination in commercial poultry production, the negative results found in flock I cannot be attributed only to the introduction of non-infected chicks.However, the results suggest that obtaining Salmonella free-birds is the first step for preventing Salmonella on the farm.

Flocks Number of analyzed eggs Number of positive eggs Positive eggs (%) Serovars
Salmonella serotypes other than those isolated from the transport boxes were detected during rearing and production in the four positive flocks.Besides vertical transmission, Salmonella may be introduced in poultry farms by several ways, mainly by feed containing meal of animal origin (Berchieri Júnior et al., 1993).Rodents have great importance on the epidemiology of avian salmonellosis, because they maintain paratyphoid salmonellas such as serotypes Enteritidis and Typhimurium in the poultry houses (Davies & Wray, 1995a).According to these authors, even after cleaning and disinfection, poultry houses are re-contaminated by infected resident rodents.Furthermore, the deficiency on the disinfecting process may contribute to the maintenance of Salmonella at farms (Davies & Wray, 1995b).It should be also considered that light lines of birds are supposed to be resistant to Salmonella.In fact, the hens did not show signs of illness although they were infected and bacteria were shed for some time.Therefore, although natural and individual resistance of birds might be considered (Protais et al., 1996;Duchet-Suchaux et al., 1997;Bumstead, 2000), it is necessary to remember that while commercial lines of chickens are homogeneous for production, there is not enough knowledge about their susceptibility to infection.Moreover, genes responsible for the resistance to systemic infection are not related to resistance to the colonization of the intestinal tract by Salmonella (Bumstead, 2000).
Several factors might be related to the non-isolation of Salmonella sp from fecal samples in flocks B and M during the first production cycle and the absence of infection in Flock I. Birds acquire natural resistance against enteric pathogens with the gradual development of the intestinal flora and the immune system (Suzuki, 1994;Gast, 1997).In addition, an appropriate cleaning and disinfection program associated with a good quality of living birds might decrease the risk of environmental contamination (Barrow, 1999).
Molting is a debilitating process used to extend the productive life of the birds.During molting, birds become more susceptible to infection by pathogens.In fact, flocks A, B and M were evaluated after molting (76 weeks-old) and were excreting Salmonella, similar to what was reported by Nakamura et al. (1994), Holt (1995), and Macri et al.(1997).
The percentage of contaminated eggs was 0.2 and 2.0% in Flocks A and L, values in accordance to the values of 0.1 to 10.0% reported by Humphrey (1994).Although Salmonella was detected in few eggs, inadequate storage conditions and food prepared with raw eggs can be a serious threat to human health (Humphrey et al., 1989).Eggs may become contaminated by Salmonella in the ovary and oviduct (Thiagarajan et al., 1994;Keller et al., 1995;Miyamoto et al., 1997).Most of the times, however, contamination occurs in the cloaca during egg passage.It is well known that the number of Salmonellacontaminated eggs decreases, together with a decrease in fecal shedding.
Most food-borne infections caused by Salmonella in humans were associated to food like mayonnaise, ice cream and frozen desserts, which are consumed without being cooked after raw egg is added.Provided that few Salmonella organisms are present on egg shells or egg contents, they multiply in a few minutes during storage at room temperature (Duguid & North, 1991).
Similar to the results described by Zancan et al. (2000), SE was also predominant among the serotypes isolated from transport boxes of one-day-old chicks.This serotype was also frequently isolated from chicken feces after one week of age and was detected in eggs laid by flock A. SE has been associated to food-borne disease since the last century (Barrow, 1993).However, in the last decades, there was an increase in human cases, and most cases were associated to poultry products (Barrow, 2000).In the present work, SE was detected in chicks from flocks A from one-day old until the end of the trial, resulting in the production of contaminated eggs.Besides, SE intoxication also increased in Brazil in the last years (Taunay et al., 1996;Hofer et al, 1997) and thus, it is evident that more efficient control measures are needed.
Salmonella Infantis, also isolated from fecal samples from four flocks, was implicated in food borne human salmonellosis in Finland related to consumption of poultry meat (Nurmi & Rantala, 1973).S. Infantis and S. Mbandaka were isolated from eggs, from flies and other insects present in the chicken houses, and from swab samples taken from the environment (Jones et al., 1995;Kinde et al., 1996;Olsen & Hammack, 2000).These two serotypes were also isolated from humans presenting symptoms of salmonellosis (Scheil et al., 1998;Dera-Tomaszewska & Glosnicka, 1999).According to Hoszowski & Wasyl (2001), domestic poultry is the main source of S. Mbandaka infection for men.
Salmonella Javiana isolated from feces in Flocks B and L had already been detected in domestic chickens by Adesiyun et al. (1998).It also causes gastroenteritis in human and was considered the sixth most incident serotype in 1996 by CDC, in USA.In Brazil, according to Tavechio et al. (1996) and Villa (2000), S.Infantis, S. Mbandaka and S. Javiana are among the serotypes frequently isolated in samples from human, poultry and other animal sources, from the environment and from feed and feedstuffs.
In many occasions rough strains of Salmonella were isolated.The biochemical behavior was compatible with the profile of paratyphoid strain.Some evidence suggested they might be of the serovar Enteritidis, nevertheless, the impossibility of identifying them hinders any epidemiological analysis.
In the present experiment the presence and the persistence of Salmonella were shown in laying hen flocks since their arrival until the end of the production period.These results demonstrated that improvements are needed in Salmonella control program because those currently adopted have not been able to prevent the introduction and dissemination of salmonellas on poultry farms, as well as egg contamination.

Table 1 -
Isolation of Salmonella sp from transport boxes of one-day-old birds.
* Positive flocks used to carry on the research.**Negative flock used to carry on the research.***R: rough strain.

Table 2 -
Isolation of Salmonella sp from transport boxes and feces of laying hens.

Table 3 -
Salmonella sp serovares isolated from transport boxes and feces of light white laying hen flocks.

Table 4 -
Serovar of Salmonella sp isolated from eggs produced by 52-week-old layers.