Two groups of four calves each were immunized either intramuscularly (i.m. vaccinated) or intranasally (i.n. vaccinated) at 2 and 6 weeks of age with ca. 10 9 CFU of a derivative of P. multocida serotype B:2 strain 85020 containing a deletion in the aroA gene (strain JRMT12). Both groups of calves and three unvaccinated control calves were challenged subcutaneously at 8 weeks of age with ca. 10 7 CFU of the wild-type 85020 strain. The first and second vaccinations caused a significant pyrexia and increase in the mean demeanor score (P < 0.05) in i.m. but not i.n. vaccinated calves. Serum agglutinating activity against whole cells of P. multocida strain 85020 and immunoglobulin G antibody concentrations increased after the second vaccination in i.m. but not in i.n. vaccinated animals, and this difference was statistically significant (P < 0.05). Concentrations of serum amyloid A (SAA) increased significantly 3 h after both the primary (P < 0.05) and booster (P < 0.001) i.m. vaccinations, but not in i.n. vaccinated calves. All four i.m. vaccinated calves were solidly immune to challenge with wild-type P. multocida B:2. However, the mean rectal temperatures, demeanor scores, and serum SAA concentrations of i.n. vaccinated and control calves increased significantly (P < 0.01). Three i.n. vaccinated and two control calves were killed for humane reasons within 14 h postchallenge, and postmortem examination revealed pathological lesions consistent with hemorrhagic septicemia. These data showed that the aroA mutant strain, given i.m. as two doses 4 weeks apart, acted as an effective live-attenuated vaccine strain to protect calves against challenge with the virulent parent strain.Hemorrhagic septicemia (HS) caused by infection with Pasteurella multocida serotype B:2 is a commonly fatal systemic disease of cattle and buffaloes in countries of South and Southeast Asia (3, 22). The disease is peracute, having a short clinical course involving severe depression, pyrexia, submandibular edema, and dyspnea, followed by recumbency and death. Some control is achieved with alum-precipitated or oil adjuvant broth bacterins injected subcutaneously, but these vaccines have the disadvantage of providing only short-term immunity (4 to 6 months and up to 1 year for alum adjuvant and oil adjuvant vaccines, respectively) (21), and the high viscosity of oil adjuvant vaccines makes them unpopular among field users. The disease remains a significant obstacle to sustainable agriculture in the region, and in attempts to elicit longer-term immunity, live vaccines have been developed (14), although they have been ill defined and of questionable safety. On the other hand, a marker-free aroA deletion derivative (strain JRMT12) of a virulent field isolate of P. multocida B:2 (strain 85020) obtained from Sri Lanka has been shown to be attenuated and to confer a high degree of protection when used as a live vaccine in a mouse model of hemorrhagic septicemia (19). The construct is particularly suited to be a live vaccine candidate, as it possesses no...

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Three groups of five calves, namely, V1, V2, and V3, were immunized intramuscularly at 4 and 8 weeks of age with ca. 10 9 , 10 8 , and 10 7 CFU, respectively, of a derivative of Pasteurella multocida B:2 wild-type strain 85020 containing a deletion in the aroA gene (strain JRMT12). The first and second vaccinations resulted in significantly (P < 0.01) higher rectal temperature responses in groups V1 and V2 than in group V3. Serum immunoglobulin M (IgM) and IgG titers did not increase in any group until after the second vaccination and were then significantly higher in groups V1 and V2 than in group V3 (P ‫؍‬ 0.001 for both IgM and IgG). All vaccinated groups and three unvaccinated challenge control calves (group CC) were injected subcutaneously at 10 weeks of age with ca. 10 7 CFU of strain 85020. Vaccinated calves survived the challenge, but two CC animals developed clinical disease and were killed for humane reasons. After challenge, mean serum amyloid A concentrations were significantly higher (P < 0.001) in the CC group than in the vaccinated groups. Postmortem examination revealed that calves in the CC group showed the most extensive range of bacteriologically positive tissues and gross and histopathological lesions. Overall, a clear dose-dependent response was present, with those receiving a higher vaccine dose being less affected clinically, bacteriologically, and pathologically by the wild-type challenge. The V2 treatment appeared to give the best combination of high immune response, protection, and safety.

Nanobiosensor platforms have emerged as convenient and promising approaches with remarkable efficacy for the diagnosis of infectious diseases. Gold nanoparticles (AuNPs) have been widely used due to numerous advantageous properties such as optical, electrical, physicochemical and great biomolecules binding capabilities. This study aimed to apply AuNP-Probe Conjugate for the detection of Leishmania spp., using colorimetric and amplification methods targeting parasitic ITS2 fragment. The first method was carried out by hybridization of 10µL of DNA with 4 µL of probe and addition of 5 µL of 0.2 N HCl (non-amplification method). Second method was followed by polymerase chain reaction (PCR) amplification using thiolated primer, 5 µL of AuNP and 5 µL of 0.2 N HCl. The appearance of red and purple colors indicated positive and negative results, respectively. The minimum of detection for non-amplification and amplification methods for three strains of Leishmania namely L. major, L. tropica and L. infantum were determined to be 32 fg/µL and 16 fg/µL, respectively. Sensitivity for detection of visceral leishmaniasis (VL) for non-amplification and amplification methods included 96% and 100%, respectively and for cutaneous leishmaniasis (CL) included 98% and 100%, respectively. The results of this investigation revealed that sensitivity of amplification method was the same as RT-qPCR, while that of non-amplification method was lower. However, this method was promising because of no need for any equipment, high specificity, enough sensitivity, low cost and rapidity (less than 30 min) to complete after genomic DNA extraction.