Synergistic haemolytic activity and its correlation to phospholipase D productivity by Corynebacteruim pseudotuberculosis Egyptian isolates from sheep and buffaloes

Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).

Synergistic haemolytic activity and its correlation to phospholipase D productivity by Corynebacteruim pseudotuberculosis Egyptian isolates from sheep and buffaloes.

Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).

Humoral and cellular immune responses of a murine model against Corynebacterium pseudotuberculosis antigens.

Humoral and cellular immune responses were both found to be operative in five groups of Balb/c mice following two subcutaneous inoculations with different antigens of C. pseudotuberculosis. These antigens included toxoid, bacterin, bacterin-toxoid with and without oil adjuvant in addition to the live cell of C. pseudotuberculosis. The responses were assessed, twenty days after the 2nd immunization. Serum antibody levels were determined in an enzyme linked immunosorbent assay (ELISA). Cellular immune responses to C. pseudotuberculosis antigens were measured by detection of gamma interferon (IFN-gamma) in spleen cell culture media of the immunized mice, using commercial mice enzyme immuno-assay kit. All mice were challenged 2 weeks after the last dose of immunization with live C. pseudotuberclosis (2x10(5) CFU/mouse). Protection levels were observed with different degrees between the immunized mice groups.

Cloning and expression of recombinant MPB70 protein antigen from Mycobacterium bovis BCG for diagnosis of tuberculosis.

In a search for developing new skin test reagents, MPB70 protein antigen was a candidate antigen for the Diagnosis of bovine tuberculosis. First M. bovis BCG genomic DNA was extracted purified and the mpb70 gene was amplified by PCR. The gene was then ligated to an expression vector, PQE. After transformation of the expression E. coil M15 host strain with the PQE plasmid, the expression was induced using 10 mM of IPTG. Two bands were seen in the SDS-PAGE analysis the 44 and 50 KDa represents the dimmers of the nonglycosylated and glycosylated form of the reMPB70 antigen. The His-tagged reMPB70 antigen was then purified by metal affinity chromatography using Ni-NTA agarose. Protein refolding was done by the use of the co solvent Polyethylene glycol MW 3000. The diagnostic potential of the re-MPB70 was evaluated using sera from experimentally sensitized guinea pigs with different strains of mycobacteria (M. bovis BCG, M. tuberculosis, M. kansasii and M. intracellular) using ELISA test. The results indicated the efficiency of MPB70 but not bovine PPD to discriminate between M. bovis sensitized guinea pigs and those sensitized with other mycobacterial strains at serum dilution of 1150. In a field trials to using reMPB70 antigen for the serodiagnosis of bovine tuberculosis using ELISA test. Fifty serum samples from tuberculin +ve and 6 from tuberculin -ve cattle were used as well as 10 tuberculin +ve buffaloes. All +ve animals were confirmed to be M. bovis infected by P/M analysis, bacteriological examination. ELISA results revealed that reMPB70 could recognize the tuberculin +ve infected animals at serum dilution of 1/50 and that it could diagnose tuberculosis in cattle as well as buffaloes.

Production and evaluation of a monoclonal antibody to Clostridium perfringens type D epsilon toxin.

A monoclonal antibody (mAb) against C. perfringens type D epsilon toxin was produced in hybridoma tissue culture supernatant and in BALB/C mice ascitic fluid. The mAb reacted with single band of the purified epsilon toxin and efficiently neutralized the lethal effect of epsilon toxin in mice. A competitive ELISA (C-ELISA) was developed using this mAb and used to evaluate C. perfringens type D vaccines. Individual serum samples from vaccinated rabbits were tested using C-ELISA and mouse neutralization test (MNT). The mAb based C-ELISA correlated with the conventional MNT for estimation of antitoxin level.

Immune regulation of ovarian function in buffaloes (Bubalus bubalus).

We studied the infiltration of different subsets of immune system cells in the ovarian parenchyma of Egyptian buffaloes during follicular and luteal phases of the estrous cycle. All subsets of leukocytes infiltrated significantly more into corpora lutea (CL) than into Graafian follicles (GF) (P < 0.01) except for plasma cells that were abundant in the GF but not observed in the CL. The number of macrophages, lymphocytes, neutrophils and eosinophils were significantly greater in mature CL than in corpora hemorrhagica (CH) or regressing CL. Moreover, the regressing CL showed significantly more macrophages, lymphocytes and neutrophils than the CH. Large antral follicles were infiltrated with larger number of leukocytes than growing preantral atretic follicles. Macrophages and neutrophils observed in large antral follicles were significantly more abundant in the theca externa than the theca interna (P < 0.01). Only plasma cells were significantly greater in number in the theca intema (P < 0.01). Leukocytes infiltrated significantly more into large mature follicles than large, growing, preantral atretic follicles (P < 0.01). Results of this study reveal the calling of leukocytes in a significant numbers inside the ovarian tissue of buffaloes around the time of ovulation and at luteolysis. It is possible that leukocytes with their powerful bioactive cytokines (IL-1, TNFalpha, GM-CSF, and INF-gamma) may assist in ovarian functions such as ovulation and luteolysis.

Oedematous skin disease of buffalo in Egypt.

This review covers a historical view and etiology of oedematous skin disease which affects buffalo in Egypt, the microbiology of Corynebacterium pseudotuberculosis causing the disease: its virulence; clinical signs; mechanism of pathogenesis; histopathology; mode of transmission; immunological aspects; treatment and control. It is concluded that C. pseudotuberculosis serotype II is the main cause of OSD and exotoxin phospholipase D and its lipid contents of the cell wall are the major causes of pathogenesis. After declaring the role of Hippobosca equina in transmission of the causative agent among buffaloes, control of OSD is now available.

Number of viable bacteria and presumptive antibiotic residues in milk fed to calves on commercial dairies.

OBJECTIVE: To assess the number of bacteria and presumptive antibiotic residues in milk fed to calves and to identify those bacteria and the antibiotic susceptibility of selected bacterial strains. DESIGN: Cross-sectional prospective study. SAMPLE POPULATION: 189 samples obtained from 12 local dairies. PROCEDURE: Samples of waste milk and milk-based fluids (eg, milk replacer, colostrum, bulk-tank milk) were obtained. Cumulative number of viable bacteria was determined. Bacteria were cultured aerobically, and antibiotic susceptibility testing of selected strains was performed. Presumptive antibiotic residues were detected by use of test kits. RESULTS: Geometric mean of the cumulative number of bacteria for waste milk samples was significantly higher than for other types of milk or milk-based products. Streptococcus sp (84/165 samples) and Enterobacteriaceae (83/165 samples) were the predominant bacteria identified, followed by Staphylococcus sp (68/165 samples). Escherichia coli was the gram-negative species most commonly isolated (52/165 samples; 32%); however, none were strain O157. Salmonella sp or Mycoplasma sp were not isolated. Of 189 samples, 119 (63%) were positive when tested for beta-lactams or tetracycline by use of 2 commercially available assays. In vitro, some bacteria were resistant to commonly used antibiotics. CLINICAL IMPLICATIONS: Waste milk that has not been effectively treated (eg, pasteurization) to reduce microbial load prior to use as calf feed should be used with caution, because it may contain a high number of bacteria that may be pathogenic to cattle and human beings. Antibiotic residues that would constitute violative amounts and existence of multiple antibiotic resistant bacterial strains are concerns in calf health management and dairy food safety.

The effect of Escherichia coli J5 and modified live Salmonella dublin vaccines in artificially reared neonatal calves.

One thousand neonatal calves, allocated in a factorial design into four groups, were vaccinated subcutaneously with two doses each of either killed Escherichia coli (0111:B4) J5 bacterin or a UC Davis modified live, genetically altered (aro-) Salmonella dublin vaccine, or both, or with a placebo. In this prospective double-blind study to determine the immunogenicity and protective effects of both vaccines on bovine neonates in field conditions, calves were observed daily until 2 months of age, and serum samples from selected study calves were obtained at five different time points. No clinical adverse vaccine reactions were observed. Overall mortality was 7.5% (75 of 1000), E. coli and S. dublin infection being the most commonly associated aetiological agents of deaths. Both J5 (p < 0.01) and Salmonella (p = 0.05) vaccines were significantly effective in reducing the mortality rate but without an additive effect. The role of passive transfer was important in calf survival. The E. coli J5 and (aro-) S. dublin vaccination schedule employed significantly (p < 0.001) elevated J5 and Salmonella-specific serum ELISA antibody titres, respectively, by the sixth week of age.

Passive immunotherapy in neonatal calves--I. Safety and potency of a J5 Escherichia coli hyperimmune plasma in neonatal calves.

A pair of studies designed to assess the clinical safety and potency of hyperimmune plasma administration was undertaken in neonatal calves. Bovine plasma from Holstein heifers hyperimmunized with a mutant Escherichia coli O111:B4 (J5) vaccine which had a geometric mean (GM) immunoglobulin G (IgG) ELISA titer of 3.5 x 10(4), was administered subcutaneously to < or = 2-day-old calves. In the first (study A) of two prospective trials, hyperimmune plasma was administered in two doses to colostrum-deprived (CD) (n = 7) and colostrum-fed (CF) (n = 16) neonatal dairy calves. Data were collected immediately before (0 h) and 6, 24, 48, 72, 96, 120 and 288 h after the first plasma administration. Total serum protein and serum IgG concentration elevated in both groups with a significant (p < 0.01) rise of E. coli J5-specific IgG from the corresponding baseline by 6 h post-plasma administration. In the second (study B) trial, calves (N = 75) allocated into three protocol groups received two doses of either the hyperimmune (TT) J5 plasma (n = 30) or the control (CT) plasma (n = 30), and the third group (NT) received no plasma (n = 15). Data were collected up to 96 h post-plasma administration. Serum IgG and J5 specific ELISA antibody titer increased significantly (p < 0.01) in TT calves compared to the other group calves. The endotoxin contents of the administered plasma were < or = 1 EU ml-1 by limulus amebocyte lysate (LAL) method and the collected physiological and hematological data values were similar in all groups in both trials. In addition, no immediate adverse reaction or death was observed in any phase of the plasma administration. The E. coli J5 hyperimmune bovine plasma, as prepared and administered to neonatal calves in the current trials, proved to be a safe and potent biomedical fluid.

Passive immunotherapy in neonatal calves--II. The efficacy of a J5 Escherichia coli hyperimmune plasma as immunotherapy in neonatal calves.

Hyperimmunized bovine plasma containing antibodies to a mutant Escherichia coli O111:B4 (J5) was used to conduct a prospective double-blind clinical trial to evaluate its efficacy as an immunotherapy to bovine neonates in field conditions. Two- to three-day-old calves (N = 150) were randomized into three groups (n = 50) to receive (1) no plasma (NP) or (2) control (traces or no J5 antibody) bovine plasma (CP), or (3) hyperimmune bovine anti J5 plasma (HP) in two subcutaneous total doses of 10 ml kg-1 body weight at a 24 h interval. Various physiological, pathological and clinical parameters of the study subjects were observed up to three weeks while other data such as morbidity, mortality and the effect on heart girth increase were collected up to the end of the eighth week. Weekly serum total protein and immunoglobulin G (IgG) concentrations were preferentially increased from the baseline values in HP calves but not statistically significant (p > 0.05) in group comparison. Mean (geometric) serum J5 ELISA titers in the HP group were significantly (p < 0.001) higher than the other two groups that increased about 1-log by the first week of plasma intervention, followed by a gradual decline by the third week. Out of three total deaths due to septicemia and colitis, one was from the NP group while the other two were from the HP group. Morbidity as measured daily on a 13-point scoring scale were not statistically (p > 0.05) different among the groups. Variation in the mean heart-girth increase was non-significant (p > 0.05) among groups by the eighth week. Higher increase in heart girth was generally associated with higher initial serum IgG (p < 0.01) concentration. Our results suggest that this lot of hyperimmune J5 plasma at this dose was not superior to control plasma or to no intervention in terms of calf morbidity and mortality.

Rotavirus infection in calves in Bangladesh.

Faecal samples from 434 calves under 1 year of age (307 diarrhoeal and 127 normal) were collected from three dairy farms and one village in selected areas of Bangladesh. The samples were tested by an enzyme-linked immunosorbent assay (ELISA) to detect the presence of rotavirus antigen. Of 402 dairy calves tested, 28 (7.0%) were positive, of which 21 (7.2%) were from diarrhoeic calves and 7 (6.3%) from non-diarrhoeic calves. Rotavirus infection varied from farm to farm (2.7-9.2%) and there was no positive response from any of the 32 village calves. Rotavirus was most commonly found in calves of 1 week of age or less (up to 22.2% in one group) but was not found in any calves later than 6 months of age. More than 80% of rotavirus-positive samples from diarrhoeic calves exhibited a titre of 128 or more (geometric mean 345 +/- 4.5), whereas non-diarrhoeal calves had titres less than or equal to 128 (geometric mean = 29 +/- 1.9), suggesting that rotavirus infection in calves in Bangladesh was mostly associated with diarrhoea.

Studies on histoplasmosis farciminosi (epizootic lymphangitis) in Egypt. Isolation of Histoplasma farciminosum from cases of histoplasmosis farciminosi in horses and its morphological characteristics.

Isolation of Histoplasma farciminosum from five horses, showing typical signs of histoplasmosis farciminosi (epizootic lymphangitis) was successfully attempted. The mycelial form of H. farciminosum was isolated on Sabouraud dextrose agar enriched with 2.5% glycerol, brain heart infusion (BHI) agar enriched with 10% horse blood and PPLO dextrose glycerol agar. The last medium proved to be the most effective, both for primary isolation and subculturing of the fungus. It was found that on primary isolation, the lag phase of the mycelial form of the fungus was relatively long, involving 4-8 weeks at 25 degrees C. Colonies of the mycelial form of H. farciminosum appeared on subculture as a yellowish, light brown to deep brown, convoluted, waxy, cauliflower-like growth tending to form scant aerial growth. Conversion of the mycelial form to the yeast form of H. farciminosum was successful by subculturing either on BHI agar with 5% blood or on Pine's medium and incubating at 35-37 degrees C. Complete conversion to the yeast form was achieved only after 4-5 repeated serial transfers onto fresh media every 8 days. The yeast colonies were flat, raised, slightly or deeply wrinkled, white to light gray to grayish brown, and were pasty in consistency.

Isolation of Cryptococcus neoformans, Candida albicans and other yeasts from pigeon droppings in Egypt.

Two hundred pigeon droppings, unmixed with soil, and collected from various provinces in lower Egypt (Delta) were cultured to detect yeasts. Yeasts were isolated from 53.5% of the samples; Cryptococcus neoformans was recovered from 30 samples and Candida albicans from 19 samples. Other Cryptococcus and Candida species as well as species of Torulopsis and Rhodotorula were also encountered. Aspergillus fumigatus was isolated on 13 occasions.