Title page for ETD etd-04032007-195119


Type of Document Dissertation
Author Perry, Quinesha Laticia
Author's Email Address qperry1@lsu.edu
URN etd-04032007-195119
Title Brucella melitensis: The Evaluation of a Putative Hemagglutinin Gene's Effect on Virulence in the Caprine Model
Degree Doctor of Philosophy (Ph.D.)
Department Pathobiological Sciences (Veterinary Medical Sciences)
Advisory Committee
Advisor Name Title
Philip H. Elzer Committee Chair
Alma F. Roy Committee Member
Frederick M. Enright Committee Member
Richard K. Cooper Committee Member
Mandi J. Lopez Dean's Representative
Keywords
  • Virulence
  • Hemagglutinin
  • Brucella melitensis
  • Homologous Recombination
  • Gene Replacement
  • Caprine Model
Date of Defense 2007-03-29
Availability unrestricted
Abstract
Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortions in goats and sheep and Malta fever in humans. The zoonotic disease brucellosis causes severe economic losses in the Mediterranean region and parts of Africa, Asia, and Latin America.

With the completion of the genomic sequences of B. abortus 2308 and B. melitensis 16M, no classical virulence factors were found; and the chromosomes were virtually identical. However, in B. melitensis, a putative hemagglutinin gene was identified which is absent in B. abortus. The possibility of the hemagglutinin gene being a potential virulence factor was evaluated via gene replacement/deletion in B. melitensis and expression in trans in B. abortus.

The hemagglutinin gene was PCR-amplified, cloned into pBBR1MCS-4, and electroporated into B. abortus 2308 yielding B. abortus 2308-QAE. A kanamycin-Region E-kanamycin disrupted gene fragment (KAN-E-KAN) was also generated and electroporated into B. melitensis 16M. The resulting mutants were characterized biochemically to confirm its Brucella origin and screened by antibiotic selective pressure.

A colonization study of non-pregnant goats infected with B. abortus 2308, B. melitensis 16M, B. abortus 2308-QAE, or B. melitensis 16MÄE revealed no attenuation of the 16MÄE mutant when compared to 16M at 4, 7, and 21 days post inoculation. The study also showed that both the variant and the mutant were capable of infecting and disseminating throughout the host.

All four strains were introduced into the pregnant goat model and evaluated for pathogenicity. Pregnancy/delivery results revealed 27%, 78%, 67%, and 50% abortion rates in goats infected with 2308, 16M, 2308-QAE, and 16MÄE, respectively. Bacterial culture of tissues from 2308, 16M, 2308-QAE, 16MÄE -exposed goats revealed 45 %, 79%, 75%, and 100% colonization of dam/kid pairs, respectively. The expression of the B. melitensis 16M hemagglutinin gene in trans in 2308-QAE revealed a significant (p<0.05) increase in colonization and abortion rates when compared to B. abortus 2308, mimicking the virulence of B. melitensis 16M in pregnant goats. The B. melitensis 16MÄE disruption mutant colonization and abortion rates demonstrated no attenuation in colonization but did show a 28% reduction in abortions when compared to B. melitensis 16M.

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