Molecular Detection of Salmonella gallinarum and its Prevention using Autogenous Bacterin in White Crystal Neck Commercial Layer Birds

Muhammad Zulqarnain Shakir¹, Farzana Rizvi^1*, Muhammad Kashif Saleemi¹, Muhammad Wasim Usmani¹, Nasir Mahmood¹, Faisal Rasheed Anjum², Nouman Amjad³, Muhammad Shahzad Shafique¹ and Ayesha Ramzan^1
*Correspondence: Farzana Rizvi, Department of Pathology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan, Email:

Keywords

Salmonella gallinarum • Autogenous bacterin • Humoral immune response

Introduction

The success of the flock, growth, and welfare in commercial poultry depends upon the use of a good vaccination program. A vaccine can help to prevent or reduce the effect of harmful diseases when used in conjugation with good biosecurity and managemental conditions. Many vaccines are used to control Salmonella infection such as live vaccine may become ineffective in immunocompromised individuals and the killed vaccine are serovar specific, produces only short immunity [1]. Live and inactivated vaccines are made from rough 9R strain and have been widely used to control fowl typhoid. The number of viable organisms/doses is important to control its transmission (through eggs and from bird to bird). Vaccination with 9R produces transient antibodies and may precipitate high mortality in the infected flocks. The primary dose of the 9R vaccine is usually done at the 8^th week of age & booster dose at the 16th week of age [2]. Despite these limitations, a broad-spectrum killed bacterin will be good to control Salmonella. Bacterin is a major component of the vaccination program and is used to stimulate high-level immunity. These contain inactivated organisms suspended in aluminum hydroxide or water in oil adjuvants. They induce an immune response by producing protective antibodies, protecting the birds from harmful bacterial pathogens such as Salmonella spp., during the laying period [3]. To our knowledge, very little work has been done yet in Pakistan to characterize the non-motile poultry Salmonella at the molecular level and to prepare bacterin from a local isolate. Keeping in view the economic importance of this disease the comprehensive research will help in the formation of preventive strategies, in the long run to curtailing economic losses to the poultry industry. Therefore, the current study was designed to prepare of bacterin vaccine from local isolated S. gallinarum and its comparative efficacy study of commercially available and locally prepared bacterin against S. gallinarum

Materials and Methods

Isolation of S. gallinarum was done from cloacae swabs of birds, briefly; a sterile cotton swab was incorporated into the cloaca of clinically sick birds to collect cloacal contents. After collection, every swab was shifted to a 10 ml tube containing tetrathionate broth and incubated at 37°C overnight. A loopful of broth was streaked on SS agar for examination of S. gallinarum colonies. Suspected colonies were examined biochemically and morphologically.

Salmonella specific gene amplification by PCR

Isolated bacterial colonies were picked up and mixed with distilled water for isolation of DNA template for PCR. The Salmonella-specific set of forward and reverse SPG primers to target SG gene were used [4]. A PCR reaction mixture of 25 μl was prepared by taking 12.5 μl of Master mix, 1 μl of each primer, 2 μl of DNA template, and 8.5 μl of water in the tube. The reaction was carried out in thermocycler with 35 cycles to amplify SG gene with the following conditions: initial denaturation for 3 minutes at 95℃, denaturation at 94℃ for 20 seconds, annealing at 59℃ for 30 seconds, extension at 82℃ for 30 seconds and final extension at 72℃ for 5 minutes. The PCR product was analyzed through gel electrophoresis at 60V/cm for 50 minutes in a 1.5% agarose gel stained with 2.5 µl of ethidium bromide.

Challenge inoculum

Broth culture of S. gallinarum was taken and centrifuged at 3000 r.p.m for 10 minutes. The supernatant was collected and discarded and the remaining was mixed and diluted with buffer saline using MacFarland alike tube to have 109 CFU/ml, infected dose was prepared according to Timms LM, et al. [5].

Bacterin preparation

The autogenous bacterin was made from the locally isolated field strain of S. gallinarum following the technique of Timms LM, et al. [5]. The isolated field strain was cultured in nutrient broth followed by nutrient agar and incubated at 37℃ for 24 hours. Thereafter brain heart infusion agar was used for culturing and incubated at 37℃ for 48 hours. The growth of S. gallinarum was shifted in normal saline at room temperature for 24 hours and 1% formalin was used for inactivation. The washed concentration of inactivated bacterin was adjusted to contain 1011 CFU/ml by using MacFarland matching tubes. Sterile concentration was obtained by adding equal volumes of washed inactivated bacterin and Montanite oil and stored in the refrigerator for further use. The prepared local whole-cell deactivated bacterin was provided to layer birds at the 6th week of age and a booster dose (0.2 ml/bird) was given at the 9^th week of the period. The whole-cell deactivated bacterin was offered in two shots intramuscularly (I/M) in the thigh muscle.

Quality control test of autogenous bacterin

The formulated whole-cell deactivated bacterin was verified for purity, safety, and efficacy test according to the basic procedures described by British veterinary codes (1970).

Purity test of inactivated bacterin

A purity test was performed before the formalin deactivation of the S. gallinarum strain. This test confirms the purity of broth culture and did not contain any organism before inactivation. This purity test was done by inoculating the broth culture on SS agar and incubating it at 37℃ for 24 hours. The presence of pure colonies of S. gallinarum on SS agar confirms the purity of broth.

Safety test of prepared bacterin

Safety was done following the method of Dorsey (1963). Mice (n=10) were injected (0.2 ml of vaccine) subcutaneously (S/C). The vaccinated mice were kept under observation for 14 days to observe any clinical signs and symptoms.

Efficacy test of prepared autogenous bacterin

Layer birds (n=120) of crystal neck breed were used in this study. Day-old layer birds were purchased from a local hatchery and kept under good managemental conditions. Fresh and clean water was ensured, and commercial Mukhtar feed of layer was given throughout the experimental trial. The layer birds were kept under understudy for 15th weeks of age.

Experimental design to check the efficacy of bacterin vaccine

One hundred- and thirty-layer birds, day-old were purchased and divided into three groups having 40 birds in each. Upon arrival, ten birds were slaughtered and observed bacteriologically to ensure Salmonella's free flock. The layer chicks were divided as follows.

Group A: Control negative (Non infected and non-treated)

Group B: Control positive (S. gallinarum infected birds)

Group C: Vaccinated and S. gallinarum infected birds

Parameters measured during experimental trial for the efficacy of bacterin

Birds were monitored for clinical signs, mortality, and morbidity rate for three weeks post-infection. Deceased birds were exposed to postmortem assessment for reflection of gross and histopathological study.

Detection of shedding of bacteria

To ensure birds were free from S. gallinarum infection before the experimental trial, the cloacal swabs were taken from each group.

Subsequently the challenge, swabs were taken from infected as well as a control group on weekly basis and assessed bacteriologically to check shedding of S. gallinarum infection. The cloacal contents were collected using the cloacal swab, swab was inserted and rotated clockwise to collect contents. After which the collected swabs were transferred to tetrathionate broth for the enrichment and incubated at 37℃ for 24 hours. Colonies were studied morphologically and bacteriologically.

Upon arrival, the birds were weighed and weekly production parameters such as body weight and Feed Conversion Ratio (FCR) were measured.

Humoral immune response

Just before 1^st vaccinal dose, the 10 birds were selected randomly and from the wing vein blood samples were taken. Also, before the booster dose, the 10 birds were chosen haphazardly from each group and blood samples were taken from the same marked birds. On weekly basis, the blood was collected from each group post-infection and sera were collected. The collected sera were stored at -20℃ for further use.

The antibody titer against S. gallinarum was determined through ELISA according Muktaruzzaman M, et al. [6], and the Microagglutination test according to Brown, etc., 1981. In MAT the antibody was expressed as Geometric Mean Titer (GMT), while in ELISA the antibody titer was assessed through S/P ratio by using the following formula:

S/P ratio: Sample mean- Negative control/ positive control- negative control

Calculation of Antibody Titer: Log10 Titer=1.13Log ^(SP) +3.156.

Anti-Log= Antibody titer

Histopathological examination

The formalin-fixed tissue samples of the liver, kidney, spleen, intestine and heart were collected aseptically. The collected tissue samples were processed using the paraffine embedding technique. Tissue sections of 5 µ thickness were cut using the microtome followed by hematoxylin and eosin staining and analyzed under a microscope to check changes.

Results

Results of quality control of autogenous bacterin

The locally prepared bacterin of S. gallinarum proved to be pure, safe, and free from any adverse side effects on crystal neck white commercial layer birds and weekly body weight gain. The affected mice did not show any clinical signs and symptoms.

Results of protective efficacy of autogenous bacterin

The locally prepared autogenous bacterin of S. gallinarum showed an 82% protection rate against wild type S. gallinarum strain in group C while in the positive control group (B) the survival rate was only 22% as shown in Table 1.

Results of shedding of bacteria (S. gallinarum) from vaccinated and challenged layer birds

The rei-solation of S. gallinarum from positive control (Group B) was 55.88%, 41.17%, 18.18%, and 9.09% at 1st, 2nd, 3rd, and 4th-week post-infection respectively compared to 15.21%, 12.19%, 4.87%, and 0% in those vaccinated with the locally prepared autogenous vaccine (Group C) as shown in Table 2.

Results of clinical signs and gross pathology

The layer birds vaccinated with autogenous bacterin showed mild clinical signs with a slight gross lesion in the intestine (enteritis). The layer birds of a positive control group (B) showed perfused white watery diarrhea, depression and birds were hesitant to move. On postmortem examination, the positive control group showed bronze discoloration of the liver having white necrotic foci, swelling of the spleen and liver. In a few birds, the pericardium was wrapped with yellowish necrotic material.

Results of Microagglutination test for measurement of antibody

The GMT of layer birds vaccinated with autogenous bacterin of S. gallinarum increased from (0) pre-vaccination to 64 at the 3rd-week post-primary vaccination and 173 after the 3rd week of a booster vaccination. After the challenge, the geometric mean titer of antibody had increased in group C (vaccinated and infected) from 173 to 275 against S. gallinarum infection while it was 65 in the positive control group (B) at 3rd-week post-challenge (Table 3).

Results of ELISA of autogenous bacterin

At 3^rd-week post-primary vaccination the ELISA antibody titer in group C (vaccinated and infected) was 850.3 against S. gallinarum infection. Moreover, a gradual increase in antibody titer was shown in group C at 3^rd-week post booster vaccination, it was 2258.7. After 3^rd week of the challenge, the antibody titer in the vaccinated group (C) had increased up to 2272.4 against S. gallinarum infection (Table 4).

On the other hand, a sudden increase of antibody titer was noted in the control unvaccinated group (Group B), where the antibody titer was 159.2 at the 3rd-week post of primary vaccination, 209.6 at 3rd-week post booster vaccination, and 898.4 at 3rd-week post-challenge (Table 4).

Molecular confirmation of Salmonella gallinarum

Conventionally confirmed isolates of S. gallinarum were confirmed by PCR targeting SG gene. All the tested isolates were successfully amplified targeting SG gene (Figures 1 and 2).

Discussion

Salmonellae gallinarum is responsible for major economic losses in poultry in terms of mortality and decrease in egg production. In our study the tested organism (S. gallinarum) was gram-negative, rod-shaped occurred singly or in paired. Small-round and black-centered colonies on XLD; pink, transparent, smooth raised colonies on MacConkey agar; black-colored colonies due to production of H2S on SS agar were observed. The isolate fermented maltose, dextrose, and mannitol produced both acid and gas, which were in line with the previous findings [7].

In the next step, the PCR-confirmed S. gallinarum isolates were used for the preparation of bacterin followed by determination of immunogenicity potential of the bacterin in layer birds challenged with S. gallinarum. Our results of the challenge protection study showed that the birds immunized with autogenous bacterin (C) showed significant protection to S. gallinarum infection as compared to the birds of the positive control (B). Only mild or no clinical signs of fowl typhoid were observed in autogenous bacterin (C). Similar findings regarding the protective potential of S. gallinarum-bacterin had also been reported by the previous reports of several studies [8]. It is summarized that locally autogenous bacterin of fowl typhoid and probiotics are effective in reducing clinical signs, mortality rate, and gross lesions in S. gallinarum infection, similarly, the protective effect of formalin killed bacterin previously reported by [9,10]. Typical clinical signs of fowl typhoid such as fever, anorexia, ruffled feathers, diarrhea, and anemia were observed in the positive control group (B). Similar clinical signs were reported previously by Lopes PD, et al. [11], Freitas Neto OC, et al. [12], Ezema WS, et al. [13], Garcia KO, et al. [14] and Shivaprasad HL [15]. An incubation of seven days was observed in our study which was in contrast to the previous findings of [14] in which three days of incubation had been reported. This variation in the disease incubation period could be associated with several factors including the infective dose, virulence, host pathogenicity, and immune response to fight against pathogenic organisms like S. gallinarum by Lahiri A, et al. [16]. A mortality and survival rate of 18% and 82% respectively was observed in birds administered with autogenous bacterin (C) in comparison to the positive control (B) in which 78% mortality and 22% survival rate was observed. These results suggest that autogenous bacterin could be a potential candidate as a prophylactic vaccine. Our results of immunogenicity potential of autogenous bacterin were following the previous studies [17-19].

In the present study, the protective efficacy of locally prepared autogenous bacterin was more as compared to the positive control group (B) and these results are in agreement with Penha RAC, et al. [20]. Fecal shedding of Salmonella gallinarum organisms in the group of chickens (vaccinated with locally prepared bacterin) reached 4.87% which was lowered than the positive control group (Salmonella infected group) similar results of fecal shedding was reported by El-Enbaawy MI, et al. [21].

Conclusion

The protection with local prepared autogenous bacterin to be safe and help the birds with fast recovery from Salmonella gallinarum infection. It helps to prevent the birds from infection with increased FCR, decreased clinical signs, and mortality. It enhances the humoral immune response in vaccinated birds as compared to non-vaccinated birds.

Conflicts of Interest

The author declares no conflict of interest as there are no direct financial or other connections with other people or organizations or that can inappropriately influence our work.

Acknowledgments

We would like to thank all participants. The authors would like to thank Dr. Farzana rizvi for assisting with project layout and Statistical analysis.

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