Rapid and visual detection of Shiga-toxigenic Escherichia coli (STEC) in carabeef meat harnessing loop-mediated isothermal amplification (LAMP).

Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94 fg/µLand 25.8 fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12 h enrichment. The study highlights the importance of short (6-12 h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9 h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

Evaluation of in-feed supplementation of formic acid and thymol as non-antibiotic growth promoters and assessing their effect on antimicrobial resistant E.coli isolated in Turkey.

With the rampant usage of antibiotics as growth promoters (AGPs) in poultry sector, there has been alarming concerns of antimicrobial resistant microbes such as Escherichia coli. Diversification of poultry farming due to consumer demand for safer products with higher protein content, turkey production is gaining popularity. Feed additives such as formic acid (FA) and thymol (TH) are effectively replacing AGPs due to their antimicrobial action. This directed the researchers to find alternatives to antibiotics such as thymol and formic acid because of their strong antimicrobial, anti-oxidative, digestive-stimulating properties. To assess the efficacy of FA and TH as growth promoters and their effect on the antimicrobial resistance (AMR) load, the current study (0-12 weeks) was conducted in CARI VIRAT turkey poults (n = 256; unsexed) those were randomly distributed into eight treatment groups: control(T1), AGP (T2), graded levels of FA (T3 to T5) @ 2.5, 5 and 7.5 ml/kg and TH (T6 to T8) @ 120, 240 and 350 mg/kg. Cloacal swab samples were collected at 0, 4th, 8th and 12th week interval and processed further for isolation, identification and assessment of resistance profile of E. coli. The final body weight, cumulative gain and FCR were significantly (p < 0.05) better for birds under supplementation. The Total plate count (TPC) and coliforms showcased a significant (p < 0.001) reduction in the FA and TH supplement groups as compared to control and AGP group. The resistance profile indicated E. coli isolates from AGP group with significantly (p < 0.001) highest resistivity against antibiotics (viz. chloramphenicol, tetracycline, nalidixic acid, chlortetracycline) while isolates from FA (T5) and TH (T8) groups were the least resistant. blaAmpC gene was significantly (p < 0.001) harbored in T2 isolates whereas least detected in T5 and T8. It was inferred that formic acid (7.5 ml/kg) and thymol (360 mg/kg) can effectively replace AGPs and lower AMR burden in poultry.

Role of narL gene in the pathogenesis of Salmonella Typhimurium.

Salmonella Typhimurium (STM) is a facultative anaerobe and one of the causative agents of nontyphoidal salmonellosis (NTS). Its anaerobic metabolism is enabled under the hypoxic environment that is encountered inside macrophages and the gut lumen of the host. In both of these niches, free radicals and oxidative intermediates are released by neutrophils as an inflammatory response. These chemical species further undergo reactions to produce nitrate, which is preferably taken up by STM as an electron acceptor in the absence of oxygen. NarL, the response regulator of the two-component regulatory system NarX/L, and a transcription factor, gets activated under anaerobic nitrate-rich conditions and upregulates the nitrate reduction during anaerobic respiration of STM. To understand the role of NarL in the pathogenesis of STM, we generated a narL-knockout (STM:ΔnarL) as well as a narL-complemented strain of STM. Anaerobically, the mutant displayed no growth defect but a significant attenuation in the swimming (26%) and swarming (61%) motility, and biofilm-forming ability (73%) in vitro, while these morphotypes got rescued upon genetic complementation. We also observed a downregulation in the expression of genes associated with nitrate reduction (narG) and biofilm formation (csgA and csgD) in anaerobically grown STM:ΔnarL. As compared with wild STM, narL mutant exhibited a threefold reduction in the intracellular replication in both intestinal epithelial cells (INT- 407) and monocyte-derived macrophages of poultry origin. Further, in vivo competitive assay in the liver and spleen of the murine model showed a competitive index of 0.48 ± 0.58 and 0.403668 ± 0.32, respectively, for STM:ΔnarL.

Isolation and characterization of vB_SenS_Ib_psk2 bacteriophage against drug-resistant Salmonella enterica serovar Kentucky.

Salmonella enterica serovar Kentucky is one of the food-borne zoonotic pathogens which is isolated in high frequency from poultry meat in the recent decades and is known for its multidrug resistance. The current study was aimed to isolate and characterize a bacteriophage against S. enterica serovar Kentucky isolate, 5925, which showed resistance to at least seven antibiotics and to study its efficiency to decontaminate S. Kentucky from chicken skin. The bacteriophage against S. enterica serovar Kentucky was isolated and was named vB_SenS_Ib_psk2 representing the place, source, and host. Electron microscopy revealed that the phage possesses isometric head and contractile tail, indicative of Siphoviridae family. Molecular detection of major capsid protein E gene yielded 511 bp, and NCBI blast analysis revealed that the phage belonged to the genus chivirus. The optimum temperature and pH for phage survival and multiplication were found to be - 20 to 42 °C and 6-10, respectively. One-step growth curve experiment of vB_SenS_Ib_psk2 revealed a latent period of 20 min and burst size of 253 phages/bacterial cell. The host susceptibility studies revealed that 83% of MDR isolates of S. enterica were susceptible to vB_SenS_Ib_psk2. Artificial spiking studies on chicken skin revealed that high multiplicity of infection (MOI) of phages of 106 pfu/mL is required for significant reduction (p ≤ 0.01) of bacterial concentration (0.14 ± 0.04) after 24-h incubation at 8 °C compared to group 1 (2.55 ± 0.89 cfu/mL).

Hematological and serum biochemical parameters and profiling of cytokine genes in lumpy skin disease in Vrindavani cattle.

Lumpy skin disease (LSD) is a notifiable re-emerging transboundary viral disease of bovines that inflicts heavy losses in affected livestock farms. Genetic variations contribute substantially to the inter-individual differences in the immune response against disease agents. The present study aimed to evaluate the genetic basis of differential immune response in Vrindavani cattle by comparing the hematological, biochemical and cytokine genes' expression profiles of LSD-affected and unaffected animals. After 21 days of the outbreak at the farm, the animals were grouped as affected (those who developed symptoms) and unaffected/healthy (those who did not). Standard hematological and biochemical parameters were evaluated in both the groups. Expression profiling of important Th1 (IL2, INFG and GMCSF) and Th2 (IL4, IL6 and IL10) cytokines was also performed via a relative quantification approach using real-time PCR. Erythrogram and leucogram analyses revealed significant differences in total leucocyte count (TLC: 14.18 ± 0.74 versus 11.38 ± 0.68 x103/µL), hemoglobin (Hb: 8.66 ± 0.42 versus 10.84 ± 0.17 g%) and percentage of neutrophils (46.40 ± 1.98 versus 35.40 ± 2.11%), lymphocytes (49.40 ± 1.99 versus 62.40 ± 1.86) and monocytes (4.20 ± 0.37 versus 2.40 ± 0.40) between the affected and healthy animals, respectively. The production of liver enzymes (SGOT and SGPT) was significantly higher in affected animals (74.18 ± 4.76 and 59.51 ± 2.75) when compared to the healthy counterparts (65.95 ± 9.18 and 39.21 ± 3.31). The expression profiling of Th1 and Th2 cytokines revealed significant differences between the two groups, except IL10. The expression of IL2, GMCSF and IL6 were upregulated in healthy animals while that of INFG, IL4 and IL10 were upregulated in LSD-affected animals. The highest abundance was observed for IL2 transcripts in healthy animals among all assessed cytokines with log2fold change of 1.61 as compared to affected counterparts. Overall, the immune response in healthy animals (after exposure to LSD virus) was predominated by the expression of Th1 cell proliferation and there was an increased production of pro-inflammatory cytokines as compared to the affected counterparts. The results revealed that the effective immune response to LSD in cattle consists of changes in hematological and biochemical parameters and altered expression profile of cytokines with enhanced phagocytosis and lymphocyte recruitment. Furthermore, optimal expression of Th1 cytokines is required for maintaining optimal health against infectious insult with LSD virus in cattle.

LSDV126 gene based molecular assays for specific detection and characterization of emerging Lumpy Skin Disease virus.

Lumpy skin disease (LSD) is a highly infectious and economically important viral disease, which is currently emerging in the Indian subcontinent. LSD is caused by Lumpy Skin Disease Virus (LSDV) under the genus Capripoxvirus and the family Poxviridae. Since its first incursion in India in the year 2019, the virus is rapidly disseminating through different means like direct contact, fomites and mainly by blood-feeding insects. As the disease has never been reported from India or neighbouring countries, there is a lack of planning and preparatory measures in terms of diagnostics and vaccines to control the disease. In the absence of any homologous vaccine, a live attenuated heterologous goat pox vaccine (Uttarkashi strain) is now being widely used in the country for the prevention of LSDV infection. Use of live attenuated goat pox virus vaccine necessitates the availability of an assay which could specifically detect and differentiate LSDV from goat pox virus. In this study, nucleotide sequences of LSDV126 gene encoding extracellular enveloped virus protein of circulating LSDV and goat pox virus were determined and analyzed. Deletion of 27 nt tandem repeats was observed in LSDV in comparison to goat pox and LSDV vaccine viruses. The deletion region was targeted for designing primers specific to LSDV, but not goat pox virus. A novel isothermal polymerase spiral reaction (PSR) was optimized as pen side diagnostic for prompt and sensitive detection of genomic DNA of LSDV. The assay was found to be highly sensitive and specific when compared to the real-time PCR. The assay was found to be specifically detecting only LSDV but not the goat pox virus. The limit of detection was identified as 9 × 10-6 ng of positive DNA. The assay will provide a point of care tool that will be a boon for the successful control of LSD in India.

In vitro antioxidant and antimicrobial activities of clove extract and its effectiveness in bio-composite film on storage stability of goat meat balls.

The aim of this study was to find out clove extract's antimicrobial and antioxidant properties, as well as its efficacy as a bioactive ingredient in the development of bio-composite films to increase the storage stability of goat meat balls stored at 4 ± 1°C. The clove extracts (CLEs) were prepared in ethanol, hydroethanol (1:1), and water and evaluated for antioxidant and antimicrobial potential. In vitro assays of CLEs revealed more susceptibility for gram-positive bacteria than gram-negative bacteria. Among the different extracts, the clove ethanol extract (CLEE) had the highest antimicrobial activity against tested microorganisms as well as total phenolics (1.14 mg GAE/g), flavonoids (8.50 µg catechin/g), and DPPH assay (39.59%). Further, the concentration-dependent effect of CLEE (p < 0.05) on thickness and color values and antimicrobial properties of the bio-composite film were observed. The storage qualities of the product T1 (with film; 450 µl CLEE) such as pH (6.45 ± 0.01), TBARS (0.87 ± 0.06 mg malonaldehyde/kg) value, free fatty acid (0.193 ± 0.001% oleic acid), total mesophilic count (4.98 ± 0.05 log10 CFU/g), and sensory attributes (overall acceptability score: 5.67 on 8-point scale) were better (p < 0.05) than T0 (without film; control) on day 20 of storage. Thus, the ethanolic clove extract has a superior antioxidant and antimicrobial potential. Its inclusion in the bio-composite film prolonged the storage stability of goat meat balls by controlling lipid oxidation and microbial growth. Practical Application Today's consumers are more attracted towards meat products added with natural ingredients having preservative effects. Clove extract is a classic example of a natural preservative and has excellent antimicrobial and antioxidant potential. The present study revealed that by wrapping the ethanolic clove extract-based bio-composite film on goat meat balls extended the storage stability of the product due to controlled lipid oxidation and microbial growth. Thus, such bio-composite films can be successfully applied on goat meat balls that function as a antimicrobial packaging for providing optimum organoleptic quality and better shelf life.

Antimicrobial resistance in Indian isolates of non typhoidal Salmonella of livestock, poultry and environmental origin from 1990 to 2017.

A retrospective antimicrobial resistance study of nontyphoidal Salmonella enterica isolates from India during 1990-2017 was conducted to study the microbial susceptibility to antibiotics. A total of 271 Salmonella enterica isolates from poultry (n = 146), farm animals (n = 55) and environmental sources (n = 70) were tested for susceptibility using 15 antimicrobial drugs. The drug classes include aminoglycosides, phenicols, cephalosporins, penicillins, carbapenems, fluoroquinolones, and sulphonamide-trimethoprim. Study revealed that overall, 133 (49.08%) of 271 isolates were resistant to ≥ 1 antimicrobial drugs and 81 (29.89%) out of 271 isolates were multidrug resistant (resistance to ≥ 3 drugs). Majority (68.96%) of Typhimurium serovars (n = 87) were susceptible to all antibiotics tested, whereas only 5% Kentucky serovars (n = 40) were pan susceptible. All the drugs revealed decreasing trend of susceptibility from 1990 towards 2017 except cephalosporins and carbapenems. Statistical analysis of association between time period and antimicrobial resistance revealed a significance of < 0.05. Molecular detection of genetic determinants associated with antimicrobial resistance revealed the presence of genes like class I integrons, sul1, sul2, catIII, cmlA, dfrA, blaTEM, blaAmpC in the resistant isolates. Furthermore, plasmid mediated quinolone resistant determinants like qnrD and qnrS were also reported in the current study.

Development of BruAb2_0168 based isothermal polymerase spiral reaction assay for specific detection of Brucella abortus in clinical samples.

Bovine brucellosis, predominantly caused by Brucella abortus is one of the most neglected zoonotic diseases causing severe economic losses in the dairy industry. The early and precise diagnosis of the disease is required to reduce the transmission of infection in humans as well as animals. In the current study, a rapid and novel isothermal amplification-based polymerase spiral reaction (PSR) was developed for the specific detection of Brucella abortus by targeting the BruAb2_0168 gene. The assay could be conducted at 65 °C in a water bath and results can be obtained after 60 min. The detection limit of the PSR assay was found to be 1.33 fg. The sensitivity of the assay was found to be 104 fold higher than conventional PCR and equivalent to real-time PCR (RT-PCR). The assay didn't exhibit cross-reaction with selected pathogenic non-Brucella bacteria and Brucella spp. other than B. abortus. Forty clinical samples were also tested using this novel assay and it was able to detect 25 samples as positive, however, conventional PCR could detect the targeted organism in 22 samples only. To the extent of our knowledge, this is the first report towards the development of a PSR assay for specific detection of B. abortus. The assay can be used as a quick, sensitive and accurate test for the diagnosis of bovine brucellosis in the field setting. Relatively one of the paradigm-shifting aspects of this assay would be it does not require any expensive equipment and the results can be easily visualized by the unaided eye, therefore making PSR a valuable diagnostic tool in field conditions.

Sero-epidemiology of porcine parvovirus, circovirus, and classical swine fever virus infections in India.

Reproductive problems in swine caused by porcine viruses pose a serious threat to the pig industry in developing countries like India. For evaluating the true extent of porcine infections, a total of 1308 representative sera samples were collected from 92 different pig farms covering 8 North-Eastern states and Punjab state of Northern India during a period of 2 years (2011-2013). Sera samples were tested for the presence of antibodies against porcine parvovirus (PPV), porcine circovirus-2 (PCV-2), and classical swine fever virus (CSFV) using commercial enzyme-linked immunosorbent assay (ELISA) kits. In the North-Eastern states, the seroprevalence of CSFV in non-vaccinated animals was 6.30% and that of PCV2 and PPV was 6.28% and 1.24%, respectively. In Punjab, the seroprevalence of CSFV in non-vaccinated animals was 44.44% and seroprevalence of PCV-2 and PPV was 34.07% and 39.10%, respectively. Detection of antibodies against more than one virus revealed that 4.66% animals had co-infection with PCV-2 and PPV, 1.75% with CSF and PPV, 1.98% with CSF and PCV-2, and 1.75% with all the three viruses. The receiver operator characteristics (ROC) curve analysis depicted that piglet mortality, parvovirus, and CSFV were the most important parameters with an AUC value of 0.997, 0.897, and 0.973, respectively. Incidence of single or co-infection with different viruses showed that the occurrence of single infection was significantly more prevalent than co-infection. This study provides useful information to set up future epidemiologic, flock management, and public animal health policies for the prevention and control of PCV-2, PPV, and CSF in India.

Optimization of ingredients for preparation of low calorie fiber enriched chhana balls-Sandesh like product.

The present study aimed to optimize the relative proportion of roasted flax seed flour as dietary fiber ingredient (DFI) and jaggery:stevia percent in preparation of chhana balls. Response surface methodology (RSM) was used to determine the optimum DFI proportion and jaggery:stevia percent. Thirteen experimental runs were conducted with varying levels of independent variables viz. dietary fiber ingredient (4.0-8.0%) and jaggery:stevia :: 1:1 (22.5-27.5%), as generated by central composite design. The responses investigated were pH, cooking yield, water activity (aw), Hue angle, Chroma value and sensory attributes of chhana balls. The RSM results showed that the experimental data could be adequately fitted to a second-order polynomial model with a satisfactory Coefficient of determination (R2 > 50%). The study revealed that the effect of all the factors were significant on the studied responses. The optimum formulation obtained using desirability function was 5.92 and 26.42% for DFI proportion and jaggery:stevia respectively. The values of responses at optimum formulation were 6.36 pH, 91.80 cooking yield (%), 0.9481 water activity (aw), - 22.62 Hue angle, 8.71 Chroma value, 6.89 sweetness and 7.10 overall acceptability. These predicted values were validated with experimental values and found be not significantly different.

Development and evaluation of isothermal amplification assay for the rapid and sensitive detection of Clostridium perfringens from chevon.

Loop-mediated isothermal amplification (LAMP) is a promising, simple, rapid and sensitive molecular detection method. In the present study, LAMP assay was developed for detecting Clostridium perfringens in chevon. Primers were designed to detect the cpa gene of C. perfringens. A panel of 19 bacterial strains, including 3 C. perfringens and 16 other strains, were included in this study to standardize and evaluate the LAMP assay. No false positive amplification was observed indicating 100% specificity of the assay. The detection limit of LAMP and conventional PCR in the DNA extracted from pure C. perfringens was 0.34 pg and 3.4 pg, respectively. This revealed that LAMP assay is 10 times more sensitive than conventional PCR. The sensitivity of the LAMP assay for the detection of C. perfringens in raw chevon was found to be 1.2 × 102 CFU/g after 6-h enrichment and 1.2 × 105 CFU/g without enrichment in artificial spiking studies. Improved C. perfringens detection of 12 CFU/g within 12 h was obtained proving that LAMP assay is significantly faster than traditional methods that take >2 d. The developed LAMP assay also detected the targeted organism in clinical and environmental samples with the sensitivity and specificity of 97% and 84%, respectively with Kappa agreement of 0.824 respects to PCR assay. This method shows immense potential for routine diagnosis and monitoring of C. perfringens in food, environment and clinical samples. This is the first report in which the LAMP assay was optimized for the detection of C. perfringens in chevon.

Comparative diversity analysis of ruminal methanogens in Murrah buffaloes (Bubalus bubalis) in four states of North India.

We compared the community structure of methanogens in Murrah breed of buffaloes of four states of north India using 16S rRNA gene clone library method. The results revealed the dominance of methanogens related to Methanobrevibacter in three states, while Methanomicrobium-related methanogens were abundant in one state.

Anti-oxidant and anti-microbial properties of mutton nuggets incorporated with blends of essential oils.

The present study was carried out to evaluate effect of natural anti-oxidants on the quality of mutton nuggets. Different blends of essential oil were evaluated for incorporation in mutton nuggets and it was found that Blend-1 had significantly higher sensory scores. Then, Blend-1 was tried at 0.25, 0.5 and 0.75% levels and product containing 0.25% level received significantly higher sensory scores. Thereafter, two combinations of flaxseed flour and 0.25% Blend-1 were tried viz., 4% flaxseed flour + 0.25% Blend-1 and 8% flaxseed flour + 0.25% Blend-1. Evaluation of sensory and physico-chemical properties were done in mutton nuggets incorporated with 0.25% Blend-1 (T-1) and selected combination (4% flaxseed flour + 0.25% Blend-1) (T-2). T-2 had significantly higher dietary fiber and crude fiber than T-1 products. These products were then assessed for quality changes during storage at refrigerated temperature for 30 days at 5 days interval. Significantly lower TBARS values were recorded for treatment products than control at each interval of storage period. T-2 product showed significantly higher DPPH value than other products. Microbial count remained within the permissible limit of log104 cfu/g for TPC, PC, yeast and mould count up to 15th day, 25th day and 30th day for control, T-1 and T-2 products, respectively. Essential oil and their combination incorporated mutton nuggets had about 10 days longer shelf life than control.

Development and Evaluation of Simple Dot-Blot Assays for Rapid Detection of Staphylococcal Enterotoxin-A in Food.

The present study was aimed to develop and evaluate dot-blot assays for rapid detection of staphylococcal enterotoxin-A (SEA) in food. Dot blots were developed in two formats, indirect and sandwich utilizing mouse monoclonal anti-SEA and rabbit polyclonal anti-SEA antibodies. In indirect dot-blot format, recombinant SEA was directly coated on NCM dot-blot strip and detection was carried out by anti-SEA antibodies. In sandwich dot-blot format, SEA was trapped between anti-SEA capture and detection antibodies. Both the dot-blot assays exhibited a sensitivity of ~48 ng ml-1 when tested in different food matrices. The developed assays were highly specific as no cross-reactivity was detected with other classical staphylococcal enterotoxins, toxigenic bacteria and foodborne pathogens. Sensitivity and specificity of developed indirect and sandwich dot-blot assays with respect to PCR was found to be 100 and 99%, respectively. The results shows that the developed dot-blot assays can be used as rapid preliminary screening tests for detection of SEA in food or determining the toxigenic potential of staphylococci, especially in resource-limited settings.

Proteomic Characterization of Lytic Bacteriophages of Staphylococcus aureus Isolated from Sewage Affluent of India.

Staphylococcus aureus is a Gram-positive bacterium that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to need for an alternative treatment such as bacteriophage therapy. Present study describes isolation and characterization of a staphylococcal phage from sewage samples. S. aureus isolates obtained from microbial type culture collection (MTCC), Chandigarh, India, were used to screen staphylococcal phages. A phage designated as ΦMSP was isolated from sewage samples by soft agar overlay method. It produced clear plaques on tryptone soya agar overlaid with S. aureus. Transmission electron microscopy revealed that the phage had an icosahedral symmetry. It had 5 major proteins and possessed a peptidoglycan hydrolase corresponding to 70 kDa. ΦMSP infection induced 26 proteins to be uniquely expressed in S. aureus. This phage can be proposed as a candidate phage to treat staphylococcal infections.

Comparative analysis of protein profiles of wild virulent (E156) and aroA-htrA double deletion mutant vaccine strain (S30) of Salmonella enterica subsp. enterica serovar Abortusequi under in vivo and in vitro growth conditions.

In the present study, cell lysate and cell supernatant of the both strains i.e., virulent wild type (E156) and mutant (S30) vaccine strains of Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi), grown under varied in vivo and in vitro conditions were subjected to SDS PAGE and western blotting (using rabbit hyperimmune serum). Variation in growth conditions did not have any significant effect on expression of different proteins. SDS PAGE of E156 and S30 cell lysate (CL) revealed 26 and 28 bands, respectively with 3 prominent proteins of 71, 46 and 42 kDa in cell lysate of E 156 and 4 prominent proteins 71, 65, 46 and 40 kDa in S30 strain. The cell supernatant (CS) from both the strains, subjected to SDS PAGE, exhibited similarity in protein profile among these strains, however three bands of 65, 53 and 40 kDa were more prominent in CS preparation of S30, whereas a 56 kDa protein was prominent in CS of E156. Western blotting of E156 and S30 revealed 3 unique proteins of 65, 53 and 40 kDa present in CS preparation of S30 strains which could be used for differentiation of mutant and wild strains and also in development of test for differentiating vaccinated animals from naturally infected.

Antigenic competition among different 'O' antigens of Salmonella enterica subspecies enterica serovars during hyperimmunization in pony mares.

The present study on antigenic competition among somatic 'O' antigens of different Salmonella groups (A, B, C1, C2, D and E1) in mares revealed that the immune response to most of the antigens was not (A, B, C2) or little (C1, D) affected by antigenic competition. However, E1 group antigen, which induced high antibody titres (Avg. 12967.3) when given alone, produced almost 3.5 log2 lower antibody titres on giving with other antigens, indicating the antigenic competition among some Salmonella group antigens. The antigenic competition varied for different antigens even of the similar chemical nature. Therefore, antigens belonging to different somatic groups should not be given together for the purpose of raising polyvalent serum or for immunization using multivalent Salmonella vaccines prepared from strains of different 'O' groups revealing antigenic competition.

Novel haemolysins of Salmonella enterica spp. enterica serovar Gallinarum.

Haemolysins of Salmonella are important due to their probable role in pathogenesis of systemic salmonellosis and use in sub-serovar level typing. The present study was undertaken to determine haemolytic potential of Salmonella Gallinarum strains through phenotypic and genotypic methods. Amplification of haemolysin gene (clyA) and cytolysin gene (slyA) was attempted in order to determine their role in haemolysin production. Study on 94 strains of S. Gallinarum revealed the production of two types of haemolysis viz., beneath the colony haemolysis (BCH) or contact haemolysis and clear zone haemolysis (CZH). Haemolysis was observed on blood agar prepared with blood of cattle, buffalo, sheep, goat, horse, rabbit, guinea pig, fowl, and human blood group A, B, AB and O. Although, haemolysis was also observed on blood agar prepared with whole blood, clarity of zone was more evident on blood agar made from washed erythrocytes. Clear zone haemolysis was best observed on blood agar prepared with washed erythrocytes of goat and a total of 12% (11 of 94) S. Gallinarum strains under study produced CZH on it. The clyA gene could not be detected in any of the 94 strains under study, while slyA gene could be amplified uniformly irrespective of haemolytic potential (CZH) and haemolytic pattern (BCH) of the strains. The study suggested that the two types of haemolysis (CZH and BCH) observed among S. Gallinarum strains may not be due to either slyA or clyA gene products and thus there may be some other gene responsible for haemolytic trait in Gallinarum serovar. Different haemolytic patterns of strains under study indicated multiplicity of haemolysins in S. Gallinarum.