Embrionated eggs of laying hens experimentally infected with Salmonella gallinarum

Introduction

Salmonellosis is a disease produced by the enterobacteria of the Salmonella genus. The serovar specific to birds is Salmonella Gallinarum which has two biotypes: S. gallinarum and S. pullorum, described as etiological agents of fowl typhoid and pullorum disease respectively (Colusi et al., 1984; Shivaprasad, 2003).
Salmonella gallinarum subsists in commercial farms in Latinamerica (Chacana & Terzolo, 2003). Thus, in Argentina after the implementation of the national plan for poultry health, the National Health and Food Quality Service (SENASA) ranked it among the diseases that require routine monitoring at growers facilities due to the significant dissemination rate through vertical transmission (SENASA, 2004). Although through this type of route, the number of infected eggs only accounts for 3%, newborn chicks act as vectors and multipliers of the disease. On the other hand, the horizontal transmission also occurs when healthy birds get the infection through contaminated feces and through cannibalism at farms (Chacana & Terzolo, 2003). In the poultry industry, control and prevention of typhoid produced by Salmonella Gallinarum, should not be neglected, due to its impact at epidemiological and productive levels. That is why, the objective of this paper is to study the SG infection of embryonated eggs from laying hens, in order to make some prevention and control proposals.

Materials and Methods

Embryonated Eggs
Ninety Salmonella-free embryonated eggs from commercial laying hens were used. They were incubated in an Argentina-made Yonar electronic incubation equipment, model 300AD with automatic egg turning every sixty (60) minutes, at 36,5 ºC and 70 % relative humidity. The test was carried out at the poultry facility of the experimental field of Luján University.

Inoculation
For the infection, 11-day embryos were inoculated through the allantoic cavity, (accounting for vertical infection) with 1 x 10 colony forming unit (CFU) per egg. In order to do so, ninety eggs were observed under the ovoscope but only forty five of them were infected resulting in two treatment arms: control and infected. An area of the allantoic membrane was selected, far away from the embryo and free of blood vessels on the lateral aspect of the egg and it was disinfected with alcohol. A tuberculin syringe was introduced in order to inoculate 0,1 ml of a SG-containing solution, without tearing the internal membrane and sealing it off with a liquid adhesive.
The species used was SG INTA 91 (donated by Dr. Horacio Terzolo from the Agricultural Experimental Station of the Balcarce National Agriculture and Livestock Technology Institute, INTA-EEA Balcarce, Argentina).

Experimental design
The trial included 2 treatments, with 45 repetitions randomly distributed.
T1: Treatment with no inoculation (Control) T2: Treatment inoculated with SG.

Sampling
In order to confirm the presence or absence, samples of the liver, gut, heart and yolk sac were taken at the embryo's 14 days of age and the recount was made only on the yolk sac. Eighteen (18) days later, the sampling was taken again excluding the stomach and spleen.
Upon birth, the following samplings were added: fecal matter, caecal union, kidney, lung and cecum. For each sampling, ten chicks were used randomly.

SG Detection
The methodology to detect and isolate the SG consisted of placing the samples in 10 ml of buffered peptone water (BPW) and incubating them at 37°C for 24± 2h. Later, 0,1ml of BPW was transferred to 10 ml of Rapapport-Vassiliadis soya broth (RVS), for enrichment and incubated for 24 ±1 h at 42°C. Afterwards, the solution was planted on a selective and differential medium, Xylose Desoxicolate Agar (XLD), which was incubated for 24±1h at 37°C. For the quantification of salmonellas in the birds, 1g of fecal matter was placed in 10 ml of BPW and shaken vigorously for 30 seconds and planted on the surface of the selective and differential medium. The same procedure was carried out for the Salmonella recount in the embryo annexes.

Results

After 21 days of incubation 40 chicks hatched out from the control treatment and 32 from the infected arm with a 17.78% difference between them. Mortality was seen among infected embryos at all incubation stages and there was a 6.67% of unborn pecked embryos (Table 1).

Table 1. Percentage of embryo mortality during incubation

Treatment	Mortality until day 18	Mortality after day 18	Unborn
Control	0	11,11	0,00
Infected	6,67	15,56	6,67

Only in one embryo the bacteria was not isolated from any of the samples after 14 days of incubation. The SC recount in embryo annexes was only possible in 4 embryos. SG presence was observed in a large amount of organs (Table 2).

Table 2. Recounts and isolates of SG form different organs after day 14

Birds	Recount Embryo annexes	Stomach	Liver	Intestine	Heart	Embryo annexes
1	0	1	1	0	0	1
2	0	0	0	0	0	1
3	0	0	0	0	0	0
4	0	1	0	1	0	1
5	0	1	0	0	0	1
6	0	1	1	1	1	1
7	2x10³	1	1	1	1	1
8	1,2x10⁴	1	1	1	1	1
9	6,5x10⁶	1	1	1	1	1
10	3x10⁵	1	1	1	1	1

^{References: 0 absence of SG. 1 presence of SG.}

After 18 days of incubation, as was observed on day 14, only in one embryo the bacteria was not isolated from any of the samples. The recount in embryo annexes was 1x10³ to 5x10⁵ CFUs. However, in one embryo SG was isolated from embryo annexes despite the recount was negative. Fifty percent (50 % ) of animals had isolates from the stomach and intestine and no isolates from the liver, spleen and heart were found in any of them. (Table 3).

Table 3. Recounts and isolates of SG from different organs after 18 days

Birds	Recount Embryo annexes	Stomach	Liver	Intestine	Heart	Spleen	Embryo annexes
1	0	1	0	0	0	0	1
2	2x10⁵	0	0	1	0	0	1
3	4x10³	0	0	1	0	0	1
4	5x10⁵	1	0	0	0	0	1
5	1x10³	0	0	0	0	0	1
6	0	0	0	0	0	0	0
7	1x10³	1	0	1	0	0	1
8	2x10⁵	1	0	0	0	0	1
9	1x10³	1	0	1	0	0	1
10	1x10⁴	0	0	1	0	0	1

^{References: 0 absence of SG. 1 presence of SG.}

Upon hatching out, all infected embryos had SC in embryo annexes, but count was only possible with the methodology used in two of the birds with values of 5x10⁸ y 3x10⁴ CFUs.

A high percentage (80%) was detected in fecal matter, while in the other organs, the virus was isolated with ranges from 50 % to 70 % (Table 4).

Table 4. Recounts and isolates of SG in the different organs after 21 days

Birds	Recount Embryo Annexes	EA	S	L	I	H	SP	FM	CU	K	LU	CE
1	0	1	0	1	1	0	1	1	1	0	1	1
2	0	1	1	1	0	1	1	1	1	1	1	1
3	0	1	1	0	1	1	0	1	1	1	1	0
4	0	1	1	0	1	1	0	1	1	0	0	1
5	0	1	1	0	1	1	1	1	1	1	1	1
6	5x108	1	0	1	1	1	1	0	0	1	1	0
7	0	1	0	1	1	1	1	0	0	1	1	0
8	3x104	1	0	1	1	1	1	1	0	0	1	1
9	0	1	1	1	0	0	0	1	0	0	0	0
10	0	1	0	0	0	0	0	1	1	1	0	0

^{References: 0 absence of SG. 1 presence of SG. EA: Embryo Annexes; S: Stomach; L: liver; I: intestine; H: Heart; Sp: Spleen; FM: fecal matter; CU: Caecal union: K: Kidney; LU: Lung; CE: Caecum.}

Conclusions

We concluded that there was a significant percentage of embryos that died during incubation. A low SG concentration was enough to develop the infection, which seems to account for the above embryo mortality, and for the unborn pecked embryos. At 21 days, an elevated percentage of SG clearance through fecal matter was evident and isolation was present in 100% of the embryo annexes, while its presence in the various organs was higher than 50%. These results are different from those at younger ages, where among other organs, the isolation of embryo annexes was 90%.
Routine screening for SG in embryo annexes will allow an early detection of the bacteria at incubation facilities.

Bibliography

Chacana PA & Terzolo HR. 2003. Revisión sobre Pullorosis y Tifosis Aviar. Nuevos Enfoques para Viejos Conceptos. Rev. Med. Vet.84:14-20.

Colusi A, Romano R & Manetti J. 1984. Tifosis Aviaria en la República Argentina. Sanidad Aviar. 2:4-14

SENASA 2004. Manual de Procedimientos Operativos. Plan Nacional de Sanidad Avícola. Programa de Control de la Micoplasmosis y Salmonelosis de las aves. Programa de Vigilancia Epidemiología para la Influencia Aviar (SENASA, Argentina).

Shivaprasad HL. 2003. Pullorum Disease and Fowl Typhoid. pp. 568-582. In: Saif YM, Barnes HJ, Glisson JR, Fadley AM, McDougald LR, Swayne DE (Editors). Diseases of Poultry (11th ed.), Iowa State Press, Ames, IA.