Virulence Determination of Three Iranian Isolates of Salmonella Enteritidis in Day-Old Layer Chicks

Introduction

More than 2500 serotypes of paratyphoid Salmonella have been identified worldwide. Salmonella Enteritidis (SE) infection in poul- try is one  of  the  most  important  concerns  in poultry industry as well as public health (Gast, 2013; Owen, 2015; Chousalkar and Gole, 2016). This pathogen is an arbitrary intracellular pathogen that can cause local or systemic infections. It is also capable of caus- ing chronic disease without any noticeable signs. Induction of clinical  disease  depends on the bacterial strain and host characteristics such as age, genetics, route of infection, im- mune competency, etc. In a specific serotype, virulence of the  strains  can  vary  from  zero to 100%. Mortality occurs 4 to 10 days post challenge. Clinical disease  is  observed  only in the first week  of age.  In adult  birds,  SE  is colonized in the reproductive system thus vertical transmission may occur. Birds which recover from SE infection, show growth retar- dation for a few weeks (Gast, 2013).

Salmonella Enteritidis, especially phage type 4 (PT4), causes gastroenteritis in human (Chousalkar and Gole, 2016). Many disease outbreaks due to SE-infected eggs have been reported from countries such as US, UK and Germany. Recent studies in Iran have shown that SE is the predominant serotype in Irani- an poultry flocks (Morshed and Peighambari, 2010; Akbarian et al., 2012; Taheri et al., 2016; Doulatyabi et al., 2017). Virulence and pathogenicity of the relevant isolates have not been studied so far. In this study, three Iranian SE isolates from our previous investigations were compared for their virulence in day-old chicks.

Materials and Methods

Bacteria and inoculum  preparation

Three field SE isolates, designated as A20,

S32 and S34, from our Salmonella isolates collection (Morshed and Peighambari, 2010) and one S. Enteritidis PT21 (Morshed and Peighambari, 2009) were used in this study. The bacterial cultures were stored in tryptic soy broth  (TSB)  with  25% glycerol  at  -70

°C. Prior to use, a small volume of the frozen stock of each bacterial strain was streaked on the surface of a MacConkey agar plate that was incubated overnight at 37 °C (Waltman et al., 1998). The next day, one colony of each bacterial growth was used to inoculate  5 ml brain-heart infusion (BHI) broth. The BHI cultures were incubated at 37 °C for 18 h with shaking, then the cells were harvest- ed by centrifugation at 4000 x g at 4°C for  10 min, washed three times with PBS, and resuspended in PBS. One-ml samples were taken from each bacterial suspension for determination of the number of bacteria per milliliter (CFU/ml). Bacterial suspensions showed a concentration of 5×109 - 1010 CFU/ml. All media were from Merck, Ger- many.

Experiment

Fifty LSL female chicks at day  one  of age were obtained from a commercial layer breeder flock and transferred to the animal isolation facility of the Veterinary Medical Research and Teaching Hospital  (VMRTH) of the University of Tehran. On the day of ar- rival, the chicks were randomly assigned to 5 experimental groups of 10 chicks and placed in 5 well-separated battery cages. One cage (control group) was kept in a separate room. All chicks were kept under routine condi- tions and had free access to feed and water. Feed samples from supplier breeder flock  and meconium of chicks were examined  to be free from Salmonella (Waltman et al., 1998).  Feeds  for  experimental  chicks were

Reza Tavayef et al.                                                                         Iranian Journal of Veterinary Medicine

obtained from a feed mill as pellet form and were also examined to be free from Salmo- nella (Waltman et al., 1998). All negative samples were run for delayed secondary en- richment test. The five experimental groups were  assigned  as 1 to 5. Group  1  (control)

were not challenged but groups 2, 3, 4, and  5 were challenged with SE strains A20, S32, S34, and PT21, respectively, at day one of age.

For groups 2 to 5, a small catheter was used to inoculate each chick directly into the crop with 0.5 ml of assigned bacterial sus- pension. At days 2, 4, 6, and 9 post challenge (PC), two chicks from each of five groups were randomly selected, euthanized by cer- vical dislocation, necropsied, and sampled for bacterio- and histopathologic examina- tions. At day 14 PC  (end  of  experiment), all remaining chicks in all groups were eu- thanized and treated in the same manner as previous ones. Liver, jejunum, and ceca of each necropsied chick were removed asep- tically. Fecal content of jejunum and ceca was washed with sterile PBS and discarded. Then, parts of liver, jejunum, and ceca were cut and sent for histopathologic examination. The rest of liver and ceca belonging to two chicks were separately pooled and homoge- nized for serial dilution in PBS. A volume of

0.1 ml of each diluted sample was cultured on XLD agar to determine the number of colonized bacteria in the above-mentioned organs. Suspected colonies were examined using biochemical tests (triple sugar iron and urea) for Salmonella  identification.

Results

Clinical observations

In this experiment, only one chick from group 4 (S34) died four days PC. All chicks in challenged groups showed mild to    inter-

mediate diarrhea from day 2 to day 9 PC. These chicks were not alert and conscious as much as chicks in control group during the experiment. Challenged chicks appeared  to be healthy at day 14 (end of the experiment) but compared to those of control group, they had lower weight.

Bacteriologic examination

No bacteria were isolated  from  chicks in control group throughout the experiment (Table 1). Two days PC, the highest number of SE was found in liver of groups 3 (S32) and 5 (PT21) and in ceca of group 5 (PT21). At day 4 PC, load of SE in all liver samples decreased significantly. The number of bac- teria in cecal samples was also reduced but not as much as that of in livers. Ceca of the chicks from group 3 (S32) showed more se- vere infection compared to that of in other groups. At day 6 PC, bacterial counts in both liver and cecal samples showed a reduction compared to those at day 4  PC. At day 9 PC, the reduction trend in bacterial count in all examined tissues was slowly continued. At day 14 PC, most of the liver and cecal samples also showed a decrease in bacterial counts. The reduction trend was especially noticeable in liver samples and it appeared that the livers will be cleared from SE faster. Gross and histopathological examinations At necropsy, no lesions were found in chicks of control groups but all chicks in challenged groups showed enteritis and pale foci on liver until 4 days PC. Some birds in groups 2, 3, and 4 were somehow emaciated 4 days PC but not at 6 days PC. Unabsorbed yolk  sac and typhilitis  were  observed more

or less in birds of challenged groups.

In histopathology, all samples prepared from control chicks were normal but samples from challenged birds demonstrated variable lesion scores (Tables  2, 3 and 4) during   the

Table 1. Bacteriological results of liver and cecum colony counts after challenge.

experiment. Two days PC, liver samples  of all challenged groups showed moderate to severe hyperemia in sinusoids and around central veins of sinusoids. Jejunal samples of challenged groups seemed to be normal but some heterophil infiltration and epithelial hyperplasia were found in some samples. All cecal samples from challenged-birds sam- ples demonstrated inflammatory cell infiltra- tion. In groups 3 (S32) and 5 (PT21), epithe- lial necrosis was obvious in cecal  samples. At day 4 PC, livers of challenged birds were moderate to severe hyperemic together with increasing inflammatory cells in the figure of typhoid nodule. In liver samples of group 5 (PT21), bacteria were observed  in abscess- es (liquification necrosis). In all jejunal and cecal samples, heterophil infiltration and ep- ithelial necrosis were occurred. However, je- junal samples from group 4 (S34) appeared  to be normal. At day 6 PC, liver samples of challenged birds were hyperemic with fi- brotic tissue around the sinosuids. Jejunal  and  cecal  samples  also  showed  inflamma-

tory cell infiltration with epithelial necrosis. Jejunal samples from group 5 (PT21) were normal but in other groups showed inflam- matory cell infiltration together with epithe- lium and submucosal glands necrosis. At day 9 PC, liver and cecal samples did not show significant change in lesion score. Although, damage and necrosis of epithelial cells were decreased and crypts appeared to be more hyperplastic. At day 14 PC, majority of liv- ers from challenged groups were normal but a few samples were involved with mild to moderate hyperemia. Jejunal samples were also normal but some showed hyperplasia. Necrosis and destruction of epithelium to- gether with inflammatory cells infiltration were observed in the submocusa and lamina propria of all chicks in challenged groups. In general, lesion scores of liver, jejunum and cecum in histopathology, except in a few occasions, did not differ significantly (P ≤ 0.05) among four Salmonella strains A20, S32, S34 and PT21 of this study.

  Table 2. Liver lesions score. Differences in lowercase superscript letters in each column indicate statistical significance (P ≤ 0.05). DPC=Days post challenge, 0= normal, 1= mild, 2= moderate, 3= intensive.

Group/ DPC

Typhoid nodules                    Diffuse leukocyte infiltration

Score of liver parameters

+ Fibrosis

Biliary hyperplasia                             Necrosis                            Sinusoidal congestion

2        4          6           9          14        2         4          6        9       14       2        4        6        9       14       2        4        6        9       14       2        4        6        9 14

1 )Ctrl(                0       0.5       0ac                0         0.5        0         0          0        0        0        0        0        0        0        0        0        0        0        0        0        0       0.5     0.5      0b 1

2 (A2O)              0        0        1.5        0.5          0         0        1.5       0.5     0.5     0.5       0        0        0      0.5       0        0        0        0        0        0       0.5       2       1.5       1 0.5

3 (S32)               0        1         0a                 1           1         0        0.5        0        1        0        0        0        0      0.5       0        0       0.5       0        0        0       2.5     1.5       1      1.5a 2

Group/

Table 3. Jejunum lesions Score. Differences in lowercase superscript letters in each column indicate statistical significance (P ≤ 0.05). DPC=Days post challenge, 0= normal, 1= mild, 2= moderate, 3= intensive.

Epithelial hyperplasia                    Leukocyte infiltration to lamina propria                       Epithelial necrosis                               Glandular epithelial necrosis

Group/ DPC

Table 4. Cecum lesion score. Differences in lowercase superscript letters in each column indicate statistical significance  (P ≤ 0.05). DPC=Days post challenge, 0= normal, 1= mild, 2= moderate, 3= intensive.

Epithelial hyperplasia                    leukocyte infiltration to lamina propria                        Epithelial necrosis                               Glandular epithelial necrosis

2            4           6            9          14           2           4            6           9           14           2           4            6           9           14          2            4           6            9 14

                                           
  1 )Ctrl(              0           0            0            0           0          1.5         1.5         0b                1            0           0            0           0            0                         0           0            0            0           0.5       0c

2 (A2O)            2           0            0            0           3          1.5          3           3a                 3          1.5          0          1.5         1.5        1.5                          0           0            1            2.5        1          0.5ac

Discussion

This study demonstrated the relative viru- lence of three Iranian field SE isolates (A20, S32, S34) and the known strain of SE PT21 for day-old layer chicks.

As it was expected, the peak of SE recov- ery was seen 48 h post challenge (PC) in all groups which is compatible with Bohez et al.’s (2006) trial results. In our study, day-old chicks infected with a high dose of wild type SE strain demonstrated an efficient initial colonization of the ceca 2 days post-chal- lenge (PC). These results are in accordance with results of Morgan  et al (2004),  Bohez et al. (2006), Rychlik et al. (2014) and Bar- bosa et al. (2017) indicating the SE ability   to colonize the ceca of chickens in the first days PC. In chickens, the ceca is the main colonization site for  Salmonella  (Desmidt  et al., 1996;  Rychlik  et  al.,  2014;  Moreau et al., 2016). Differences in virulence be- tween SE isolates have also been reported (Gast, and Benson, 1995; Ben Salem et al., 2017(. These researchers also reported that laying-type single comb white leghorns are more sensitive than broiler-type white plym- outh rocks to the virulence of various phage types of SE. However, in another study, broiler chicks experimentally infected with the specific phage types experienced slightly higher mortality (Dhillon et al., 1999). Pre- vious reports (Shivaprasad, 1990; Barrow, 1991; Gast, and Benson, 1995) indicate vari- ation in the virulence between the isolates of different SE phage types.

Following ingestion, SE localize in the intestine and then enter the blood stream, giving rise to bacteremia that results in hep- atitis, splenitis and omphalitis together with reduced body weight gains even though no mortality may be seen in chicks inoculated with SE as we observed in our study   (Dhil-

lon et al., 1999; Alisantosa et al., 2000). At the end of this study, chicks in control group (uninoculated) had approximately 15% more average body weight than those of challenged group. These findings were comparable with those of Dhillon et al (1999) on commercial broiler chicks. They concluded that subclini- cal infections following Salmonella outbreak in young broiler chicks or pullets are prob- ably responsible for reduced body weight gains and lack of uniformity in broilers.

In the present study, we recovered SE from all samples. Gast and Holt (1998) reported that liver and spleen were  usu-  ally cleared within 8 weeks after inocula- tion. Guillot et al (1995) and Barbosa et al (2017) indicated that there is a difference in the frequency of Salmonella colonization in the spleen and liver but not in the cecum. Asheg et al. (2001) showed the difference  in the frequency of colonization in the ce- cum and liver in the low and high dose challenges. They reported rapid  clearance of SE challenged groups (high and low dos- es), was observed at 28 DPC. Several re- searchers have shown the rapid elimination of Salmonella in birds infected experimen- tally (Humphrey et al., 1989; Timoney  et al., 1989; Barbosa et al., 2017). In contrast, SE was isolated from the visceral organs of naturally infected birds several months af- ter presumed outbreak (Chart et al., 1990; Rychlik et al., 2014; Kogut et al., 2016). In this study, all cecum samples showed large decline in bacterial cecal count from 9 DPC to 14 DPC. The susceptibility of chicks to persistent intestinal colonization and organ invasion by Salmonella decreases sharply during the 1st week after hatch (Gast and Beard, 1990; Gorham  et  al.,  1991;  Foley et al., 2013). In the present study, all liver samples  also  demonstrated  a  gradual   de-

crease in count from 2 days to 14 DPC. Some researchers have reported that  colo-

nization of organs with Salmonella might be merely dependent on host factors (Brownell et al., 1970; Fanelli et al., 1971; Barrow et  al., 1988). Also, Asheg et al. (2003) and Up- adhyaya et al. (2013) showed that ability of SE to adhere and colonize to the intestinal tract can be dose dependent. Differences in lesions might be due to differences in experi- mental design or differences between strains or SE phage types (Poppe et al., 1993).

As it has been demonstrated in Table 1, considerable decrease in colony count in all groups for liver and cecal samples are seen. Chicks hatch with immature T  lympho- cytes and become fully responsive when the chicks are around 4 days  old. Therefore,  it  is likely that cellular immunity and mucosal responses in the gut would develop signifi- cantly only after approximately 4 days of age, which would leave the chick relatively unprotected for the first few days of its life (Milanez et al., 2018).

Knowing phage types of the trial isolates cannot help us to determine its invasiveness. Several researchers reported that the differ- ence in virulence of the two SE PT4 strains in their test, showed that phage type alone is not the most important criterion for virulence (Poppe et al., 1993; Raspoet et al., 2014; and Bertelloni et al., 2017). Investigations on SE with different phage types have also noted substantial differences in virulence among those strains when inoculated to young chick- ens or adult hens by oral  route  (Humphery et al., 1989; Timoney et al., 1989; Gast and Beard, 1990(.

Histopathologic lesions due to Salmonel- la infection have been reported in various investigations (Desmidt et al., 1996; Al- isantosa  et  al.,  2000;  Rychlik  et  al., 2014;

Barbosa et al., 2017). In the present experi- mental study, samples from challenged birds showed histopathologic lesions in different visceral organs with variable lesion scores. Liver samples were hyperemic together with increasing inflammatory cells in the figure  of typhoid nodule. Liver lesions in this study were compatible with those of Desmidt et al. (1996) that reported the presence of small foci of inflammatory  cells  in the liver  from 4 days post infection. In our study, diffuse leukocyte infiltration, fibrosis, biliary hyper- plasia, necrosis and sinusoidal congestion were observed in liver samples with various severity. Similar liver lesions were described by Alisantosa et al. (2000) including acute multifocal necrosis of hepatocytes with infil- tration of heterophils. Barbosa et al. (2017) also noticed the presence of numerous small pale foci (necrotic foci) in the liver after SE infection of chicks. In the present study, his- topathologic examination of jejunum and ce- cum also demonstrated epithelial hyperpla- sia, leukocyte infiltration to lamina propria, epithelial necrosis and glandular epithelial necrosis at various degrees. Other investiga- tions have found compatible findings. Rych- lik et al. (2014) reported thickened appear- ance (hyperplasia) of ceca associated with an influx of leukocytes. Alisantosa et al. (2000) reported increased cellularity of  the  lami-  na propria of the ceca due to infiltration of heterophils and lymphocytes in the mucosa. Desmidt et al. (1996) observed many het- erophils infiltrating in the lamina propria and emigrating between epithelial cells. They re- ported granulomatous nodules in lamina pro- pria of cecum that were similar to glandular epithelial necrosis of our study. Diffuse and moderate lymphocyte infiltration in cecal lamina propria observed in this study, have also been reported by Barbosa et al.  (2017).