Salmonellosis in layer chickens : pathological features and isolation of bacteria from ovaries and inner content of laid eggs

We isolated and identified Salmonella organisms from ovaries of dead layer birds and from inner content of laid eggs of different poultry farms. The thirty eight ovarian swabs for bacteriology, visceral organs (liver, lung, spleen, egg follicles and intestine) of 38 dead birds for pathological study from 15 layer farms and 45 laid eggs (5 eggs/farm) from reported 9 Salmonella infected farms constituted samples of the study. Samples were subjected to isolation and identification of the causal agent followed by gross and histopathological study of the affected visceral organs. Seventeen of 38 ovarian swabs (44.7%) were Salmonella positive. The percentage of Salmonella Gallinarum, Salmonella Pullorum and paratyphoid causing Salmonella were 70.6%, 17.6% & 11.8%, respectively. About 53% livers of Salmonella affected birds were enlarged, congested, friable and bronze coloured with white necrotic foci. About 59% egg follicles were congested, hemorrhagic, discoloured with stalk formation and 70.6% intestines showed hemorrhagic to catarrhal enteritis. At histopathology, 76.5% livers were congested with formation of multifocal nodules and 82.4% egg follicles were congested with huge leukocytic infiltration. Infiltration of heterophils in intestinal mucosa was found in 47.1% cases. Four Salmonella isolates were found from 9 laid egg samples (5 eggs content comprised as 1 sample) and isolation rate was 44.4% reporting transovarian transmission in poultry Salmonellosis.


Introduction
Poultry is essential to the national economy of Bangladesh and the welfare of human beings.Several constraints such as the diseases, poor husbandry, low productivity and shortage of feed affect the optimal performance of this industry in Bangladesh (Haque et al., 1991).Salmonellosis in poultry causes heavy economic loss through mortality and reduced production (Khan et al., 1998).With great expansion of poultry rearing and farming, pullorum disease and fowl typhoid have become wide spread problem in Bangladesh (Rahman et al., 1997).Age wise prevalence of avian Salmonellosis showed highest infection rate in adult layers (53.25%) in comparison to brooding (14.55%), growing (16.10%) and pullet (16.10%) (Rahman et al., 2004).Salmonella are Gram negative, short plump shaped rods, non-sporeforming, non-capsulated, aerobic and facultative anaerobic organisms and classified under the family Enterobacteriaceae (OIE Manual, 2006).More than 2300 serotypes of Salmonella have been identified, only about 10% of these have been isolated from poultry (Gast, 1997).Chickens are the natural hosts for both S. Pullorum and S. Gallinarum (Snoeyenbos, 1991).Pullorum disease is usually confined to the first 2-3 weeks of age and occasionally occurs in adults (Shivaprashad, 1997).Fowl typhoid is frequently referred to as a disease of adult birds and there are also reports of high mortality in young chicks (Christensen et al., 1992).The epidemiology of fowl typhoid and pullorum disease in poultry, particularly with regard to transmission from one generation to the next are known to be closely associated with infected eggs (Wigley et al., 2001).Contaminated eggs produced by infected laying hens are thought to be one of the main sources of human infection with Salmonella Enteritidis (Humphrey et al., 1989).Eggs may become contaminated with Salmonella in two main ways: (i) Salmonella may silently infect the ovaries of apparently healthy hens and contaminate the eggs before the shells are formed.(ii) Salmonella infected bird droppings contain Salmonella that can contaminate the outer egg shells and may penetrate when crack the shell (Deryck and Pattron, 2004) Therefore, the present study was designed to isolate and identify Salmonella organism from ovaries of dead layer birds and inner content of laid eggs of different poultry farms at Mymensingh district as well as to study the pathology of different organs of Salmonella infected layer birds.

Materials and Methods
Experimental area: The study was conducted during the period from January to August 2010 at Mymensingh district.A total of 15 layer farms which contained 29,800 birds with no history of using Salmonella vaccine were selected for the study.
Isolation and identification of Salmonella organism from ovarian swabs of dead birds Collection of samples: A total of 38 ovarian swab samples were collected from the same flock along with the necropsy study.All the ovarian swabs were collected in test tubes containing 10 ml tetrathionate broth (TTB) and 200 µl of iodine-iodide solution was added in each test tube just before the collection of swab samples according to methods described elsewhere (Haider et al., 2003). .Cultural media: After overnight incubation in TTB, all the samples were primarily cultured in Nutrient agar and then subcultured in the Salmonella-Shigella (SS) agar, Triple sugar iron (TSI) agar, Brilliant green agar (BGA), Eosine methylene blue (EMB) agar (Haider et al., 2003) (Merchant and Packer, 1967;Haider et al., 2003). .Motility test: Motility test was performed for the separation of motile and non-motile Salmonella according to the method described elsewhere (Haider et al., 2003).

Pathological studies:
During the whole period of study, postmortem examinations of 38 dead birds were performed from the representative selected 15 layer farms.Gross pathological changes at necropsy were carefully observed and recorded.The tissues of different organs were preserved in 10% neutral buffered formalin for histopathological studies.The tissues were trimmed with 1.5 X 1 cm size, washed overnight in running tap water, dehydration in ascending grades of alcohol, cleaning in chloroform, blocking in paraffin and sectioned with microtome at 6 micron thickness.The sections were stained with routine hematoxylin and eosin staining (Luna, 1968) and finally the sections were studied with light microscope.

Isolation of Salmonella sp from inner content of laid eggs
This test was performed by following the method described by Poppe et al. (1992) and Haider (2009).Total 45 eggs were collected from 9 farms (5 eggs/9 farms) followed by washing and disinfection of egg surface by 70% ethanol.Manually homogenization of pooled egg contents was performed by using stirrer.Incubation of homogenized egg contents was done with pre-enrichment media (BPW) at 37 0 C temperature as a standard proportion of 1:10 fold dilution for 48 hours followed by incubation in selective enrichment media (SRV) at 37 0 C temperature as a proportion of 1:10 fold dilution for 24 hours.Then plating was performed onto selective agar media (BGA, SS, TSI & EMB agar).

Cultural prevalence with isolation and identification of Salmonella organism
In the present study, all 17 isolates of Salmonella organisms showed different cultural characteristics in different media.These were turbidity in TTB broth and isolates showed slightly yellowish white color colonies in BGA, slightly grayish color colonies in SS agar, black color colony in TSI agar, gray white colony in nutrient agar, pinkish in EMB agar and pale color colonies in Mc Conkey's agar.These findings of present study corresponded with the results of Old (1990); Yuno et al. (1995); Sharma and Katok (1996); Sujatha et al. (2003); Perez et al. (2004 ); Rybolt et al. (2005) and Ahmed et al. (2008).

Identification (Gram's staining)
In Gram's staining, all the isolates in the present investigation revealed gram-negative, rod shaped appearance and arranged in single.Chains of more than two bacilli were normally absent.Freeman (1985) described the morphology of the isolated Salmonella bacteria as small rod shaped, gram negative, single or paired in arrangement by Gram's staining which supported the findings of the present study.Similar morphological findings of Salmonella were also supported by other investigators (Islam et al., 2006;Ahmed et al., 2008).

Motility test
In motility test, 15 Salmonella isolates were identified as non-motile and 2 Salmonella were identified as motile and percentage of motile Salmonella was 11.8%.These results were similar to Christensen et al. (1996) and they found 11.69% motile Salmonella.On the other hand, there was small variation with the results of Buxton and Fraser (1977) which may be due to the difference in managemental condition of farms.The results of the present study were also supported by other investigators (Shane, 1989;Pomeroy and Nagaraja, 1991;Islam et al., 2006;Ahmed et al., 2008; who found 13.71%, 13.5%, 12.82%, and 13.5% motile Salmonella, respectively).
Histopathology: Only 17 Salmonella positive dead bird tissues of different organs were selected for histopathology.Table 2 describes the histopathological findings of different organs.In histopathological investigation, all the tissues of different organs of 17 layer birds did not evoke similar kinds of lesions.A total of 76.5% livers were congested and formed multifocal nodules with coagulation necrosis while remaining 23.5% liver showed hepatitis (Fig. 4).Besides, 76.5% lungs were severely congested and hemorrhagic and 23.5% lung showed inflammatory cells in alveoli and bronchi (Fig. 3).Infiltration of heterophils and lymphocytes in the mucosa of intestines were found in 47.1% cases.Sever lymphocytic depletion and focal necrosis in the spleen was found in 53% birds (Fig. 5).Whereas, about 82.4% egg follicles was markedly congested and showed huge leukocytic infiltration (Fig. 6).The microscopic lesions recorded in the present investigation were almost similar to the lesions described by other authors (Chishti et al., 1985;Calnek et al., 1991;Khan et al., 1998;Kinde et al., 2000;Hafeji et al., 2001;Prasanna and Paliwal, 2003;Holt et al., 2006;Msoffe et al., 2006).Briefly, in the present investigation, 44.7% Salmonella positive cases were recorded from ovarian swabs in which, Salmonella Gallinarum, Salmonella Pullorum and Paratyphoid causing Salmonella were 70.6%, 17.6% and 11.8%, respectively.Variable gross lesions were found in Salmonella affected organs of birds.A total of 53% liver, 59% egg follicles and 70.6% intestine were affected in this study.Histologically, 76.5% liver, 82.4% egg follicles and 47.1% intestines revealed tissue changes with variation in birds.Salmonella isolation rate was 44.4% in laid eggs of Salmonella-affected farms indicating transovarian transmission in poultry Salmonellosis.

Table 1. Gross pathological findings of Salmonella affected birds of different layer farms
From the above findings, it may be concluded that Salmonellosis has emerged as one of the most serious problems having adverse effects on poultry.In future for the control of Salmonella infection in poultry, vaccine production and gene level study need to be performed in Bangladesh to save the poultry industry.

. . Morphological characterization: The
presumptive colonies of Salmonella in different media were characterized microscopically using Gram's stain.

Carbohydrate fermentation test and biochemical test: Five
basic sugars such as glucose, sucrose, lactose, mannitol and maltose were used for sugar fermentation test.
. Biochemical test: It included MR test, VP test, indole test and dulcitol fermentation test

Isolation and cultural characters of Salmonella sp in inner content of laid eggs on selective media
Haider (2009)fected farms and isolation rate was 44.4%.The results corresponded with the findings ofHaider (2009)while the author reported 95% isolation rate of Salmonella organism from outer shell, 45% from inner shell, 35% from egg albumin and 50% from egg yolk.It can be concluded that, laid eggs content received contamination by Salmonella organism as a vertical transmission of Salmonellosis or/and contamination with the droppings of Salmonella infected layer birds.