Evaluation of cell-mediated immune responses and bacterial clearance in 6-10 months old water buffalo (Bubalus bubalis) experimentally vaccinated with four dosages of commercial Brucella abortus strain RB51 vaccine

Thirty water buffalo, obtained from a brucellosis-free farm, were used to evaluate cell-mediated immune responses and bacterial clearance in response to vaccination with Brucella abortus strain RB51 (RB51) in a dose-response study. The animals were randomly divided into five treatment groups. Groups I--V received the recommended dose (RD) of RB51 vaccine once, RD twice 4 weeks apart, double RD once, double RD twice 4 weeks apart and saline once, respectively. Cell-mediated immune response to RB51 was assessed by the histological examination of haematoxylin and eosin (H&E) stained sections of lymph nodes draining the sites of inoculation and by comparison of stimulation indices (SI) derived from gamma interferon (IFN-gamma) assay. A mixture of cytoplasmic proteins from B. melitensis B115 (brucellergene) was used as a specific antigenic stimulus to peripheral blood mononuclear cells (PBMC) and lymph node mononuclear cells (LNMC) up to 22 post-initial-inoculation week (PIW). Supernatants harvested at 18-24h after the in vitro antigenic stimulus were assayed for their IFN-gamma content by using a commercial sandwich enzyme-linked immunosorbent assay (ELISA) kit. Clearance of RB51 was assessed by the sequential immunohistochemical examination of sections of draining lymph nodes post-inoculation. There was no observable expansion of the deep cortex of lymph nodes on H&E sections indicating poor T-cell stimulation. All group V (control) water buffalo PBMC ELISA values were negative (SI<2.2) at all PIW sampling intervals. Overall PBMC IFN-gamma assay detected vaccinates from treatment groups' I--IV 67% (4/6), 83% (5/6), 33% (2/6) and 67% (4/6), respectively. LNMC IFN-gamma assay was unimpressive and there was a negative correlation (--.08) between the results of PBMC and LNMC of IFN-gamma assay. Clearance of RB51 occurred between 4 and 6 PIW in treatment groups I and III and between 6 and 12 PIW in groups II and IV. RB51 was not detected in any of the control animals at sampling intervals post-inoculation.     

A serological study of Babesia caballi and Theileria equi in Thoroughbreds in Trinidad

Ninety-three (93) horses were investigated for serum antibodies to Theileria equi (T. equi) and Babesia caballi (B. caballi) using the immunofluorescent antibody test (IFAT). Seventy-seven (82.8%) horses were seropositive; 31 (33.3%) were positive to T. equi compared to 64 (68.8%) to B. caballi while 18 (19.4%) horses were seropositive to both parasites. No significant differences in antibody frequencies among females and males for either T. equi or B. caballi were noted. Differences in seropositivity to B. caballi among age groups were not significant. Antibodies to T. equi were more frequent than to B.caballi in the age group 5 years and over than in the 1-2 and 2-4 years age groups (p<0.05). Unlike T. equi antibodies, B. caballi antibodies in horses in the county of Caroni were significantly less frequent when compared to other counties (p<0.05). Of 18 (19.4%) clinically ill horses, seven (42.9%) had clinicopathological evidence of anemia. Only one-third (6 of 18) horses were positive for the parasite on Wright-Giemsa stained blood smears and anemia was present in only 2. We report here that B. caballi and not T. equi may be the more common agent of piroplasmosis in Trinidad.     

Serological and bacteriological responses of water buffalo (Bubalus bubalis) vaccinated with two doses of Brucella abortus strain RB51 vaccine

Thirty-two water buffalo (Bubalus bubalis) calves aged 6?C10?months were used to evaluate serological responses to Brucella abortus strain RB51 (RB51) vaccination in a dose?Cresponse study and to compare the use of two selective media for the isolation of RB51. The animals were randomly divided into three treatment groups. Groups I?CIII received the recommended vaccine dose (RD) twice 4?weeks apart, RD twice 18?weeks apart and saline once, respectively. Lymph nodes were excised from the three groups and subjected to bacteriological examination to determine the frequency of detection of RB51. Pre- and post-vaccination blood samples were collected and tested for B. abortus antibodies using the buffered plate agglutination test (BPAT), complement fixation test (CFT), and dot-blot assay. Sera taken at all post-inoculation weeks (PIW) were negative for field strain B. abortus using the BPAT. Antibody responses to RB51 were demonstrated in all vaccinates but not in controls by CFT and dot-blot assay from 1 PIW up to 16?weeks following booster vaccination. The agreement for both assays was 80.7% and there was a linear interdependence with a Pearson??s correlation coefficient value of 0.578. The frequency of isolation of RB51 from the two selective media used was not significantly different (P?>?0.05).     

Nucleic acid distribution pattern in avian erythrocytes and mammalian lymphocytes: comparative studies by fluorescence microscopy and digital imaging analytical techniques

Nucleated erythrocytes of healthy domestic chicken and ducks, and lymphocytes of healthy Sprague Dawley rats were evaluated for nucleic acid distribution pattern, employing light and fluorescence microscopy procedures, as well as digital imaging analytical methods. The results demonstrate a unique organization of nuclear DNA of mature chicken and duck erythrocytes, as well as immature duck erythrocytes, as delineated spherical nuclear bodies that mostly corresponded with euchromatin zones of the cells in routine Wright-stain blood smears. The nuclear DNA of the rat lymphocytes, on the other hand, was observed as a more diffuse green fluorescing nuclear areas, with punctate variably-sized diffuse areas of RNA red fluorescence. RNA red color fluorescence was also evident in the narrow cytoplasm of the lymphocytes, especially in large lymphocytes, in comparison with the cytoplasm of the mature avian erythrocytes that completely lacked any nucleic acid fluorescence. Nuclear RNA fluorescence was lacking in the mature chicken erythrocytes, compared with those of the mature and immature duck erythrocytes as well as lymphocytes of both avian and rats blood. The significance of these findings lies in the establishment of normal benchmarks for the nuclear and cytoplasmic nucleic acid pattern in eukaryotic cells. These normal benchmarks become valuable in rapid diagnostic situations associated with pathologies, such as the presence of viral nuclear and cytoplasmic inclusion bodies that can alter the nucleic acid pattern of the host cells, and in conditions of cellular abnormal protein aggregations. Variability of cellular nucleic acid pattern can also aid in prognostic assessments of neoplastic conditions.     

Serologic responses, biosafety and clearance of four dosages of Brucella abortus strain RB51 in 6-10 months old water buffalo (Bubalus bubalis)

Thirty water buffalo were obtained from a brucellosis-free farm in order to evaluate antibody responses, bacterial clearance and safety to Brucella abortus strain RB51 vaccine in a dose response study. The animals were randomly divided into five treatment groups. Groups I-V received the recommended dose of RB51 vaccine (RD) once, RD twice 4 weeks apart, double RD once, double RD twice 4 weeks apart and saline once, respectively. Antibody responses to RB51 were monitored at 2, 4, 6, 8, 10, 12, 16 18, 22, 24 and 27 post-initial-inoculation weeks (PIW). Clearance of RB51 from the prescapular lymph node was evaluated at 2, 4, 6, 12, 18 and 24 PIW for groups 1, III and V and at 6, 8, 10, 16, 22 and 27 PIW for groups II and IV. To evaluate shedding of the RB51 strain, nasal, conjunctival, vaginal or preputial swabs were taken from all experimental animals at 1, 2, 3, 4, 6, 8 and 12 PIW. Sera taken at all PIW were negative for field strain B. abortus by both the buffered plate agglutination test (BPAT) and competitive enzyme-linked immunosorbent assay (c-ELISA). Antibody responses to RB51 were demonstrated in all vaccinates but not in the controls, up to 12 PIW, by complement fixation test (CFT) and the dot-blot assay with an 83.7% agreement for both tests. Clearance of RB51 occurred between 6 and 12 PIW in group I but less than 2 weeks after booster vaccinations in groups II and IV and between 4 and 6 PIW in group III. RB51 was not recovered at any time from swabs obtained from either RB51-vaccinates or non-vaccinates. The results of this study indicate that serologic responses to RB51 vaccination can be monitored by both CFT and dot-blot assay in water buffalo. Our data also indicates that RB51 vaccination does not interfere with brucellosis sero-surveillance and is safe (no serological and bacteriological evidence of spread to non-vaccinates, no adverse clinical signs or detectable abnormalities on haematology and serum biochemistry) for use in water buffalo.