Salmonella Gallinarum Research Articles

Control of Salmonella Enteritidis and Salmonella Gallinarum in birds by using live vaccine candidate containing attenuated Salmonella Gallinarum mutant strain

The ideal live vaccine to control Salmonella in commercial chicken flocks should engender protection against various strains. The purpose of the present study was to confirm the attenuation of a Salmonella Gallinarum (SG) mutant strain with deletion on genes cobS and cbiA, that are involved in the biosynthesis of cobalamin. Furthermore, evaluate its use as a live vaccine against Salmonella. For the evaluation of the vaccine efficacy, two experiments were conducted separately. Birds from a commercial brown line of chickens were used to perform challenge with SG wild type strain and birds from a commercial white line of chickens were used to perform challenge with Salmonella Enteritidis (SE) wild type strain. In both experiments, the birds were separated in three groups (A, B and C). Birds were orally vaccinated with the SG mutant as the following programme: group A, one dose at 5 days of age; group B, one dose at 5 days of age and a second dose at 25 days of age; and group C, birds were kept unvaccinated as controls. At 45 days of age, birds from all groups, including the control, were challenged orally by SG wild type (brown line) or SE wild type (white line). Lastly, another experiment was performed to evaluate the use of the SG mutant strain to prevent caecal colonization by SE wild type on 1-day-old broiler chicks. Mortality and systemic infection by SG wild type strain were assessed in brown chickens; faecal shedding and systemic infection by SE wild type were assessed in white chickens and caecal colonization was assessed in broiler chicks. Either vaccination with one or two doses of SG mutant, were capable to protect brown chickens against SG wild type. In the experiment with white chickens, only vaccination with two doses of SG mutant protected the birds against challenge with SE wild type. Although, SG mutant could not prevent caecal colonization in 1-day-old broiler chicks by the challenge strain SE wild type. Overall, the results indicated that SG mutant is a promising Salmonella live vaccine candidate that demonstrated good efficacy to control the infection by two serotypes of major importance to the poultry industry.

Experimental infection of commercial layers using a Salmonella enterica serovar Gallinarum strain: Leukogram and serum acute-phase protein concentrations

The aim of the present study was to evaluate white blood cell counts and serum protein profiles of commercial layers experimentally infected with Salmonella Gallinarum (SG) in order to better understand the pathophysiology of the disease caused by this bacterium. 180 five-day-old commercial layers were divided into 3 groups (G); G1 and G2 received 0.2 mL of inoculate containing 3.3x10(8) CFU or 3.3x10(5) CFU SG resistant to nalidix acid (Nal r)/mL, respectively, directly into their crops. G3 group did not receive the inoculum. Birds were sacrificed 24 hours before (T1) and 24 hours after the infection (T2), and three (T3), five (T4), seven (T5), and ten (T6) days after the administration of the inoculum. White blood cell counts were carried out in a Neubauer hemocytometer and in blood smears. Serum protein concentrations, including acute-phase proteins, were determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Data were submitted to analysis of variance, and means were compared by Tukey's test (P <0.05). G1 and G2 groups presented higher leukocyte counts on T4 and T5, respectively, due to the increase of circulating lymphocytes and heterophils, with a significant difference relative to G3. In electrophoresis, an increase in the serum levels of ceruloplasmin, haptoglobin, and hemopexin and a decrease in transferrin, which are acute-phase proteins, was verified. IgA serum levels did not change; however, IgG concentration increased during the infection. In conclusion, the results provide information for the better understanding of the pathophysiology of fowl typhoid.

Comparative proteomic analysis on Salmonella Gallinarum and Salmonella Enteritidis exploring proteins that may incorporate host adaptation in poultry

Comparative proteomics analysis of the cytosolic proteins of Salmonella Gallinarum (SG) and Salmonella Enteritidis (SE) isolated from poultry was performed. The constantly detected spots of serovar SG with concomitant absence in SE serovar as well as those markedly over expressed in serovar SE were selected for MALDI-TOF-MS identification. The NCBI-matched proteins that show overregulation were then further confirmed on the mRNA level by quantitative real time PCR. Identified proteins were representing diverse functional activities including energy production, metabolism, and nucleic acid synthesis. Interestingly, some recognized proteins have some relevance to bacterial virulence e.g. Salmonella pathogenicity island 1 effector protein, T-cell inhibitor protein, response regulator protein, paratose synthetase protein (RfbS) and heat shock protein 90. The study revealed the presence of some proteins of unknown function, which raise the speculation for their importance in either host adaptation or pathogenicity among SG serovars.

Infection of commercial laying hens with Salmonella Gallinarum: clinical, anatomopathological and haematological studies

This study aimed at evaluating the susceptibility of commercial laying hens to Salmonella Gallinarum (SG). Two experiments were carried using a mutant strain of Salmonella Gallinarum resistant to nalidix acid (SGNALr). In the first trial, the resistance of birds was evaluated based on clinical signs, faecal shedding, and mortality. It was carried out with six lines of commercial layers being three light white layers, considered to be resistant to SG (W1, W2, W3), and three semi-heavy brown varieties (B1, B2, B3), considered susceptible to SG. Each group contained 15 one-day-old birds. Hens were inoculated in the crop at 5 days of age with 0.2 mL of SGNALr neat culture. In addition, to each brown variety, a new group of 15 birds was challenged with 0.2mL of the same SGNALr culture diluted at 10-3. At the end of the first experiment, the surviving birds were sacrificed, and microbiological culture of liver and spleen was performed. In the second experiment, white and brown birds were inoculated with neat culture at five days of age. Samples were collected for evaluation of blood parameters and histopathology assessment at 1, 3, 5, 7, 9, 12, and 14 days post-infection. The results of the first experiment showed higher resistance of white birds (p<0.05), although there was no uniformity in the responses against fowl typhoid among the birds within these groups. In the second experiment, there were differences between white and brown birds both in blood parameters and in organ lesion intensity.

Identification of Salmonella gallinarum virulence genes in a chicken infection model using PCR-based signature-tagged mutagenesis

Salmonella gallinarum (SG) is a non-motile host-adapted salmonella that causes fowl typhoid, a severe systemic disease responsible for significant economic losses to the poultry industry worldwide. This study describes the application of a PCR-based signature-tagged mutagenesis system to identify in vivo-essential genes of SG. Ninety-six pools representing 1152 SG mutants were screened in a natural-host chicken infection model. Twenty presumptive attenuated mutants were identified and examined further. The identity of the disrupted gene in each mutant was determined by cloning of the DNA sequences adjacent to the transposon, followed by sequencing and comparison with the bacterial genome database. In vitro and in vivo competition indices were determined for each identified mutant and a total of 18 unique, attenuating gene disruptions were identified. These mutations represented six broad genomic classes: Salmonella pathogenicity island-1 (SPI-1), SPI-2, SPI-10, SPI-13, SPI-14 and non-SPI-encoded virulence genes. SPI-13 and SPI-14 are newly identified and designated in this study. Most of the genes identified in this study were not previously believed or known to play a role in the pathogenesis of SG infection in chickens. Each STM identified mutant showed competitiveness and/or virulence defects, confirmed by in vitro and in vivo assays, and challenge tests. This study should contribute to a better understanding of the pathogenic mechanisms involved in progression of disease caused by SG, and identification of novel live vaccine candidates and new potential antibiotic targets.

Control of Fowl Typhoid Using Tissue Culture Medium Waste After Harvest of Korean Wild Ginseng (Panax ginseng)

Fowl typhoid (FT), caused by Salmonella gallinarum (SG), is responsible for heavy economic losses through morbidity, mortality, and reduced egg production to the poultry industry worldwide. The use of alternative medications to control FT is important in light of emergence of multiple drug-resistant strains of SG and the public's desire to have organically grown poultry. We report the efficient utilization of tissue culture medium waste after harvest of Korean wild ginseng (TCM-KWG) (Panax ginseng) as an alternative medicine in the control of FT. In a series of experiments performed in this study, we found that oral administration of TCM-KWG through drinking water significantly reduced the mortality, organ invasiveness, and fecal shedding of SG in 1-d-old chickens experimentally infected with virulent SG. The results were positively correlated with higher dose and longer duration of pretreatment with TCM-KWG in relation to reduction in mortality caused by FT. The results of this work indicate that the TCM-KWG may be used as a cost-effective and environmentally friendly alternative medicine to control and minimize the losses due to FT in chickens.

난황 중의 항-Salmonella gallinarum 특이 항체 생성 및 가공 특성

가금티푸스 유발균인 Salmonella gallinarum 대항하는 항체의 함량이 높은 계란을 생산하기 위해 면역처리 후 산란계의 난황에서 항체를 분리하여 총 IgY와 특수 IgY의 함량의 변화와 그 특성을 조사하였다. 먼저 난황으로부터 항체의 수율과 경제적인 측면을 비교하기 위하여 난황 분리된 난황 25 g을 λ－카라기난(Sigma C-3889) 0.1%, 냉수 가용성(MSC #10387) 1%, 2%, 90℃ 가용성 (MSC #10386) 1%, 2% 및 냉수 가용성(0.1%) : 90℃ 가용성(0.1%) = 1 : 1 1%, 2%로 사용하여 분리하는 하였을 때 λ－카라기난 0.1%, 냉수 가용성 1% 비교적 높은 회수율과 total IgY, specific IgY 함량을 나타내었지만 λ－카라기난 0.1%가 더 나은 것으로 나타났다. Specific IgY는 마지막 백신 접종 후 44주간까지 꾸준히 검출되고 있는 것으로 나타났다. 산도 변화에 의한 total IgY의 잔존 함량을 보면 pH 5-9 범위에서 70% 이상 잔존하며, specific IgY도 pH 4-10에서 74% 이상 존재하여 산에 대하여 대체로 안정적임을 알 수 있었다. 열처리 후 조항체 용액을 total IgY, specific IgY 역가 높게 유지되는 것을 관찰 할 수 있었다. 이로써 난황으로부터의 면역 항체 생산의 가능성이 확인되었고 이러한 IgY는 추출과 가공처리 등에 의해 비교적 안정적인 것으로 나타나 IgY를 이용한 가금티푸스의 예방 또는 치료용의 대량 생산 가능성이 높은 것으로 사료되었다. Immunization of layers against Salmonella gallinarum(S.G.) which causes fowl typhoid resulted in production of anti-S.G. IgY rich eggs. Water soluble fraction was obtained from egg yolk using various gum solutions such as 0.1%(Sigma C-3889) λ-carrageenan; 1% and 2% cold water soluble carrageenan; 1% and 2% hot water soluble carrageenan; and 1% cold water soluble carrageenan with 1% hot water soluble carrageenan. Among them, λ-carrageenan 0.1% treatment showed a high recovery rate, possessing high IgY contents. In the range of pH 5-9, more than 70 percent of IgY was existent. Moreover, Anti-S.G. IgY was relatively heat-stable. This study revealed that immunoglobulin against fowl typhoid could be produced successfully by layers and the IgY was sustainable to further processing due to its pH and heat stability. IgY is promising to be utilized for prevention and treatment of fowl typhoid in industrial scale.

Comparison of the immune responses against Salmonella enterica serovar Gallinarum infection between naked neck chickens and a commercial chicken line

The immune responses of indigenous naked neck (NaNa and Nana) and normally feathered (nana) chickens against a Salmonella Gallinarum (SG) infection were evaluated and compared withth ose of a commercial line (B-380). Groups of 28-day-old chickens (NaNa, Nana, nana, and B-380) were immunized orally and subcutaneously with 50 μg of SG antigens. Control non-immunized animals were inoculated withsterile saline solution. All chickens were challenged with 1 LD50 of SG and mortality was recorded daily for 20 days. Antibodies to SG were measured in sera before immunization, before the challenge, 10 days after the challenge, and at sacrifice. Peripheral blood lymphocyte proliferation assays were performed using concanavalin A and SG antigens. Results showed that non-immunized Nana chickens exhibited the best natural resistance to Salmonella infection, since only 30% of them died. In contrast, all control B-380 chickens died by the 13th day. Immunization with SG induced immunity in chickens of all genotypes. Indigenous naked neck and normally feathered chickens showed a higher survival rate when compared with B-380 chickens. Immunized Nana chickens showed the highest antibody titres (P < 0.05) as well as the highest thymidine incorporation in peripheral blood lymphocytes stimulated with con A or SG antigens (P < 0.05). The results show that Nana chickens are the most resistant to SG infection and the best responders to vaccination with SG antigens.

Vaccination against Salmonella enteritidis in Dutch Commercial Layer Flocks with a Vaccine Based on a Live Salmonella gallinarum 9R Strain: Evaluation of Efficacy, Safety, and Performance of Serologic Salmonella Tests

This study describes a field trial in which 80 commercial layer flocks, with an increased risk of Salmonella enteritidis (SE) infection and placed on farms with a certified Standardized Biosecurity Programme (SBP) or a request for a SBP certificate, were vaccinated with a vaccine based on a live attenuated Salmonella gallinarum (SG) 9R strain. An evaluation is presented of the efficacy of the vaccine against SE infections, the effect on the performance of serologic Salmonella tests, and the spread of the vaccine strain to the egg content. For the efficacy study, assessment of the flock level occurrence of SE infections in the vaccinated group of 80 flocks was compared with that of a nonvaccinated group of 1854 flocks hatched in the same period. This control group was examined according to the compulsory control programme in The Netherlands. An evaluation was done of the performance of serologic Salmonella tests and the spread of the vaccine strain to the inner egg content of five of the vaccinated flocks. Findings demonstrated the flock level occurrence of SE infections in the vaccinated group (2/80 = 2.5%) to be significantly (P = 0.01) lower than that of the nonvaccinated group (214/1854 = 11.5%). Vaccination resulted in 59.0% positive test results in lipopolysaccharide BD enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against Salmonella serogroups B and D and 0% positive test results in the rapid plate agglutination test for detecting antibodies against S. pullorum (SP)/SG. The mean specificities of two blocking ELISAs (gm- and i-double antibody sandwich ELISAs) based on the flagellar antigen of SE and Salmonella typhimurium (ST) on the same sera were 99.6% and 96.1%, respectively. The vaccine strain could not be isolated from any of the 450 pools of 10 eggs. On the basis of these results, we concluded that vaccination with a vaccine based on an attenuated SG 9R strain contributes to the reduction of SE infections in commercial layer flocks. Furthermore, serologic monitoring of SE, ST, and SP/SG can still be carried out on flocks vaccinated with an attenuated SG 9R strain. Additionally, we found no indication of the spread of the vaccine strain to the egg content.

Production of formalin killed fowl typhoid vaccine using local isolates of &lt;i&gt;Salmonella&lt;/i&gt; Gallinarum in Bangladesh

A locally isolated Salmonella Gallinarum (SG) was used for fowl typhoid vaccine production. 2 x 107 colony forming unit (CFU) of SG was used for the vaccine production. The fowl typhoid vaccine was produced with the local isolates of SG (LRI 49) strain. Efficacy study of the vaccine was performed primarily in laboratory trial in 6 weeks and then in 8 weeks old commercial layers in the field. Chickens were boasted after 4 wks of vaccination. Rapid serum plate agglutination (SPA) test and ELISA were carried out for the detection of antibody response against SG vaccinated and non-vaccinated birds. Positive results of the rapid plate agglutination and the ELISA were 81% and 77% in laboratory trial, and 76.4% and 73.8% in field trial, respectively. The chickens of vaccinated and non-vaccinated groups were challenged with the infective dose (2 x 2 x 107CFU) of freshly prepared live SG bacteria and lesions were not detected in vaccinated birds at necropsy. The vaccine was proved safe and effective in terms of preventing of fowl typhoid in chickens in Bangladesh.  Key words: Fowl typhoid vaccine, potency, ELISA, plate agglutination test, Salmonella Gallinarum   DOI = 10.3329/bjvm.v5i1.1306 Bangl. J. Vet. Med. (2007). 5 (1 &amp; 2): 33-38