Isolation and characterization of bacteriophage Ib_pec2 against shigatoxigenic Escherichia coli.

This study was aimed to isolate and characterize bacteriophage against drug-resistant, shigatoxigenic Escherichia coli (STEC), one of the zoonotic, food-borne organisms associated with ruminants, mainly cattle. STEC were isolated (n = 35) from neonatal calves, dairy workers, and the surrounding environment and their antimicrobial resistance pattern was studied. Out of the 35 isolates tested, 17 isolates were found to be multidrug resistant to important antibiotics like ampicillin, amoxicillin-clavulanate, ciprofloxacin, streptomycin, and tetracycline. Bacteriophage namely Ib_pec2 was isolated against one of the STEC isolates and its morphology, genetic and proteomic characterization was done. Morphological analysis by TEM revealed bacteriophages belonging to myoviridae family. The genetic characterization of g23 gene revealed that the bacteriophage belonged to Tequatrovirus of myoviridae family. Proteomic analysis was able to identify five proteins identical to Tequatrovirus of myoviridae family. One-step growth curve experiment revealed a latency period of 40 min and a burst size of 893 pfu/bacteria. Temperature and pH ranging from 40°C to 50°C, pH 6-8, respectively. Phage could able to lyse majority of the STEC isolates. STEC are commensal organisms in the gastrointestinal tract of ruminants but are pathogenic in humans. Bacteriophages can be used as alternatives to antibiotics to control bacterial growth in ruminants and prevent its further spillage in the environment.

Evaluation of real-time PCR as an alternative for potency testing of Brucella abortus vaccines.

The aim of the research was to evaluate real-time PCR (qPCR) as an alternate method for quantitative detection of Brucella abortus strain 544 (S544) in the spleen of mice for potency testing of live B. abortus strain 19 (S19) vaccine. IS711 and eryC gene-based qPCR were optimized for calculating copy number. The copy number was further correlated with live Brucella count in the spleen by standard plate count (SPC) method. The mice were immunized with S19 and challenged with S544 on 30th Day post-immunization. The spleen of mice was collected at 15th, 21st, and 30th days post challenge (DPC) for estimation of S19 and S544 load via SPC as well as qPCR. The noteworthy difference was observed between immunized and unimmunized group by both methods at all time points. The maximum correlation between SPC and qPCR method was observed at 15th DPC in both immunized and unimmunized group. Repeated experiments at 15th DPC gave the parallel significant difference between immunized and unimmunized group by both methods. Thus novel, risk-free qPCR method can be used for the indirect culture-free potency evaluation of S19 vaccine in order to preclude the cultivation of zoonotic Brucella organisms from spleen samples.

Development of novel iron-regulated Pasteurella multocida B: 2 bacterin and refinement of vaccine quality in terms of minimum variation in particle size and distribution vis-a-vis critical level of iron in media.

To enhance the qualitative bacterial biomass per unit of media and to overcome the limitations of the existing haemorrhagic septicaemia (HS) vaccines, a comprehensive study was undertaken encompassing the role of iron on the bacterial biomass of Pasteurella multocida B: 2 to vaccine development. Trypsin digested hydrochloric acid-treated sheep blood (THSB) as a novel iron rich supplement had been devised for the first time for augmenting the qualitative bacterial biomass per unit of media which was evident with growth kinetic study. The higher recovery of iron from THSB became evident via atomic absorbance spectrophotometry. The critical level of iron in the media as well as mode of iron supplementation showed a major impact on the outer membrane protein profile of P. multocida B:2 and variation in droplet size and particle-size distribution of formulated vaccine. Immune response study against iron-regulated bacterin adjuvanted with aluminum hydroxide gel in mouse model showed that 3% THSB supplementation of casein sucrose yeast (CSY) not only augmented the growth of P. multocida B:2 significantly but conferred highest pre-challenged ELISA IgG titer and protection against pasteurellosis. Thus, THSB supplementation of CSY can resolve existing up-scaling and immunogenic potential problems of HS vaccine production.

Active immunization with Brucella abortus S19 phage lysate elicits serum IgG that protects guinea pigs against virulent B. abortus and protects mice by passive immunization.

Brucellosis is an economically important zoonosis of worldwide significance. Earlier (Jain et al., 2015) we reported methodology for generation of phage lysate preparations against Brucella abortus S19 using brucellaphage 'ϕLd'. In this study, using a fixed dose (Two mouse PD100) of lysates, the prophylactic efficacies of both plain and alum gel adjuvanted lysates were evaluated in guinea pig by direct virulent challenge and passive mouse protection test (PMPT). Strong humoral and cell mediated immune responses in guinea pigs and protection comparable to S19 vaccine was observed with low dose (1.0 µg protein and 120 µg carbohydrate adsorbed on 0.1% aluminium gel). Passive transfer of antibodies to mice using d 90 post immunization sera of guinea pig protected the animals against challenge. The study suggested the significance of humoral immunity in murine brucellosis. Further, the methodology can be explored to produce a new class of immunotherapeutic agents against bovine brucellosis.

Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544.

The prophylactic efficacies of plain and alum adsorbed lysate were evaluated by direct virulent challenge in mice model. A recently isolated brucellaphage 'ϕLd' was used for generation of lysates. Twenty four h incubated Brucella abortus S19 broth cultures standardized to contain approximately 10(8) CFU/ml were found suitable for generation of lysates. Three lysate batches produced through separate cycles did not show any significant variation with respect to protein and polysaccharide contents, endotoxin level and phage counts, indicating that compositionally stable lysate preparations can be generated through an optimized production process. Three polypeptides of ∼16, 19 and 23 kDa could be identified as immuno-dominant antigens of the lysate which induced both humoral and cell-mediated immune responses in a dose dependent manner. Results of efficacy evaluation trial confirmed dose-dependent protective potencies of lysate preparation. The lysate with an antigenic dose of 0.52 µg protein and 60 µg CHO adsorbed on aluminium gel (0.1 percent aluminium concentration) exhibited the highest protective potency which was greater than that induced by standard S19 vaccine. Phage lysate methodology provides a very viable option through which an improved immunizing preparation with all desirable traits can be developed against brucellosis, and integrated with immunization programmes in a more efficient manner.

Interaction of Antioxidant Trace Minerals Affecting Blood Picture Including Antioxidant Profile of Healthy Buffalo (Bubalus bubalis) Calves.

Copper (Cu) and selenium (Se) are antioxidants and essential trace elements that have mutual interaction and are reported to have beneficial effects at supranutritional levels. The experiment was executed to evaluate the individual impact of supranutritional levels of targeted elements with the effect of their interactions in buffalo calves. Twenty male Murrah buffalo calves of about 8-9 months (bodyweight 112.1 ± 7.69 kg) were distributed into four groups of five calves in each group and fed either a control (C) diet or supplemented with supranutritional levels of Cu (T1), Se (T2), or combination of both (T3) for 120 days. Higher (P = 0.015) values of packed cell volume were observed in group T2 at day 120; otherwise, all other hematological parameters remained comparable among groups. Over the period (day 120 vs. day 0), an enhancement in the percentage of lymphocytes (P = 0.006) with a reduction in neutrophils (P = 0.028) and hemoglobin (P = 0.024) values was observed in the control group. An enhancement in the percentage of monocytes (P = 0.031), with a reduced percentage of neutrophils (P = 0.022), was reported in groups T2 and T3, respectively. Interaction of Cu and Se at supranutritional level (T3) dramatically reduced plasma Cu (P = 0.008) level against the control values, with an improvement in Se markers (i.e., plasma Se, P = 0.041 and enzyme glutathione peroxidase, P = 0.057) over the values in calves fed supplemental Se alone (T2). Additionally, Cu (T1 and T3) was forced to decline (P < 0.05) Zn level in the plasma of buffalo calves. Cu (T1, P < 0.05) and Se (T2 and T3, P ≤ 0.01) supplementation was able to improve their respective plasma levels. The interaction of two trace elements at the supranutritional level further helped in reducing the lipid peroxidation (P = 0.01) values as well. Though antioxidant vitamins and cell-mediated immunity remained unaffected, humoral immunity against antigen P. multocida was high (P = 0.005) in the group T2. The conclusion may be drawn that supranutritional Cu and Se were capable to influence certain blood parameters with an additional interaction effect due to simultaneous supplementation in buffalo calves.

First report of whole genome sequence of septicemic Pasteurella multocida serovar B:2 'Soron' strain isolated from swine.

Pig pasteurellosis, caused by Pasteurella multocida, is an acute infection that also has economic implications for pig farmers. We report the complete genome sequence of a P. multocida, serovar B:2 'Soron' strain isolated from the blood of a pig that had died of pasteurellosis in India. The isolate was not found to be haemorrhagic septicaemia (HS) specific B:2 by the PCR assay. The genome of 'Soron' strain is a single circular chromosome of 2,272,124 base pairs in length and contains 2014 predicted coding regions, 4 ribosomal RNA operons, and 52 tRNAs. It has 1812 protein-coding genes that were also found in reference sequence PmP52Vac. Phylogenetic analysis revealed that Pm_P52VAc and P. multocida 'Soron' serovar B:2 were clustered in different clades. Pasteurella multocida 'Soron' serovar B:2 was found to cluster with the same ancestor of Pm70, which is of avian origin. The genome was found to contain regions that encode proteins which may confer resistance to various antibiotics including cephalosporin, which is used to treat pasteurellosis. The isolate was also found to harbour a phage region. This strain represents a novel multi-locus sequence type (MLST) that has not been previously identified, as all of the alleles used for MLST were found, but did not match any of the alleles in the database with 100% nucleotide identity. The most closely related ST was ST221. This is the first whole-genome sequence from P. multocida serovar B:2 of pig origin.

Selenium and copper interaction at supra-nutritional level affecting blood parameters including immune response against P. multocida antigen in Murrah buffalo (Bubalus bubalis) calves.

Minerals play important role in the diet of an animal. Bio-availability of minerals largely gets affected by absolute as well as the relative amount of each mineral present in the diet of an animal. Copper and selenium are two such an essential elements affect utilization of each other in the gastrointestinal tract. The present study elucidates the utilization of copper and selenium at supra-nutritional levels (higher than nutritional requirements). Male Murrah buffalo (Bubalus bubalis) calves (n = 10, 8-9 months, 111.7 ± 12.55 kg body weight) were divided equally into two groups and fed either a standard (Control) diet or the same diet supplemented with 0.3 ppm selenium (Se) and 10 ppm copper (Cu) (Treatment). Supplementation was made using liquid solutions of two inorganic mineral sources after mixing in the concentrate mixture and study lasts for a period of 80 days. Blood samples were collected just before starting supplementation (designated as 0 day of study) and at day 40 and 80 after starting supplementation. Blood samples were subjected to haematological parameters, plasma minerals and various oxidative stress-related parameters were determined with the cell-mediated and humoral immune response against antigen P. multocida (P52 strain). Supra-nutritional Se with Cu had higher blood monocytes (P < 0.05) and plasma selenium (P < 0.01) levels, while other hematological parameters and plasma minerals (except zinc, which was lower (P = 0.025) at day 80 in the treatment group) remained unaffected. Among markers for oxidative stress in blood, levels of lipid peroxidation were lesser (P < 0.01), at day 80 and overall mean values of the enzyme glutathione peroxidase and catalase were higher (P < 0.05) in the supra-nutritional group against control values. The overall mean activity of other oxidative stress markers including reduced glutathione, ceruloplasmin as well as the concentration of α tocopherol, retinol, and ß carotene remained unaffected due to supra-nutritional Se and Cu. Although cell-mediated immune response remained comparable (P > 0.05) between groups, higher (P < 0.05) overall mean antibody titer values, as well as the values at day 80, was reported in supra-nutritional Se + Cu group. The study concluded that supra-nutritional Se with Cu in the ration of growing Murrah buffalo calves was helpful to reduce the oxidative stress and to enhance the humoral immune response. Simultaneously, higher plasma Se level and number of monocytes in blood highlighted the additional role of selenium and copper in a ration of growing buffalo calves as compared to its normal recommended dose.

Protective role of Brucella abortus specific murine antibodies in inhibiting systemic proliferation of virulent strain 544 in mice and guinea pig.

AIM: The major objective of the investigation was to evaluate the hitherto uncharacterized potential of Brucella-specific antibodies to win the battle against virulent Brucellaabortus infection. MATERIALS AND METHODS: Brucella-specific immune serum was raised in mice. The antibody titer of serum was determined by standard tube agglutination test and indirect enzyme-linked immunosorbent assays (iELISA). Groups of mice and guinea pigs were passively immunized with serum containing specific agglutinin titers. 24 h after immunization, all animals along with unimmunized controls were challenged with B. abortus S544. Total B. abortus S544 counts in the spleen of each animal collected on the 7th day of challenge was determined to evaluate the protective index (PI) of anti-Brucella serum by statistical analysis. RESULT: A dose-dependent protective response to immune mice serum was observed in both experimental models though the values of PI of mice were higher than those obtained for guinea pigs. The PI values in mice passively immunized with 50 IU or 25 IU antibodies were 1.38 and 0.69, respectively. In guinea pigs, however, animals passively immunized with 50 IU or 25 IU antibodies showed PI values equivalent to 0.79 and 0.41, respectively. CONCLUSION: The observations support our hypothesis that the presence of antibodies inhibits the initial multiplication and eventual colonization of systemic organs by B. abortus. Therefore, a predominant antibody-mediated response induced by a vaccine is expected to protect the animal against the most severe clinical outcome of infection.

Neonatal stretched penile length: relationship with gestational maturity and anthropometric parameters at birth.

BACKGROUND: It is inappropriate to use universal cut-off points to interpret stretched penile length (SPL) measurements in newborns with variable body dimensions. AIM: To assess neonatal SPL on the basis of gestational maturity and anthropometric parameters at birth. METHODS: A cross-sectional observational study of SPL was conducted on stable newborns at a referral teaching hospital in north India between January and June 2012. Gestational maturity, SPL and anthropometric parameters (weight, length, head circumference and foot length) were recorded within 24 hours of birth. Variation of SPL in relation to gestational age and anthropometric parameters were evaluated using multiple linear regression models. The equation using lower confidence limits of 95% confidence intervals for the correlation coefficients provides cut-off points to define a small penis. RESULTS: Data from 1249 newborns demonstrated that penile growth follows the pattern of increase in body dimensions in newborns. SPL can be predicted best in relation to body and foot length taken together. CONCLUSIONS: SPL should be interpreted in relation to anthropometric parameters in newborns, particularly body and foot length.

Expression and lytic efficacy assessment of the Staphylococcus aureus phage SA4 lysin gene.

Treatment of bovine mastitis caused by Staphylococcus (S.) aureus is becoming very difficult due to the emergence of multidrug-resistant strains. Hence, the search for novel therapeutic alternatives has become of great importance. Consequently, bacteriophages and their endolysins have been identified as potential therapeutic alternatives to antibiotic therapy against S. aureus. In the present study, the gene encoding lysin (LysSA4) in S. aureus phage SA4 was cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clone revealed a single 802-bp open reading frame encoding a artial protein with a calculated mass of 30 kDa. Results of this analysis also indicated that the LysSA4 sequence shared a high homology with endolysin of the GH15 phage and other reported phages. The LysSA4 gene of the SA4 phage was subsequently expressed in Escherichia coli. Recombinant LysSA4 induced the lysis of host bacteria in a spot inoculation test, indicating that the protein was expressed and functionally active. Furthermore, recombinant lysin was found to have lytic activity, albeit a low level, against mastitogenic Staphylococcus isolates of bovine origin. Data from the current study can be used to develop therapeutic tools for treating diseases caused by drug-resistant S. aureus strains.