|Clinic:||Brigham & Women's Hospital, Harvard Medical School|
|City, State, ZIP:||Boston, MA 02115|
T.B. Hairgrove, DVM, DABVP
J.A. Thompson, DVM, DVSc, DACT, DACVPM
J. N. Rocha, Ph.D.
M. Vinacur, B.A.
C. Roberts, B.Sc.
G.B. Pier, Ph.D
1Division of Infectious Diseases, Dept Medicine, Brigham and Women's Hospital, Harvard Medical School, 181 Longwood Ave, Boston MA 02115
2Texas A&M AgriLife Extension Service, 241 D Kleberg TAMU 2471, College Station, Texas 77843-2471
3College of Veterinary Medicine and Biomedical Science, Texas A&M University College Station, Texas 77843-4475
Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomoniasis that has a major impact on production costs for beef cattle farmers. Immunization strategies to effectively protect against T. foetus are a high priority. T. foetus expresses a surface polysaccharide, beta 1-6 poly-N-acetyl glucosamine (PNAG). A PNAG-specific vaccine has demonstrated protection in pigs and horses. This study attempted to protect pregnant cows from an experimental T. foetus infection by prior vaccination with a PNAG-specific vaccine.
PNAG expression by T. foetus clinical isolates was assessed by immunofluorescence confocal microscopy using a labeled PNAG-specific monoclonal antibody. Cows were vaccinated in the neck sub-cutaneously with 200 µg of a synthetic oligosaccharide of PNAG composed of pentamers of β 1→6-linked glucosamine conjugated to tetanus toxoid (AV0328, Alopexx Vaccines) with 100 µl of Specol adjuvant, twice, 2 weeks apart. PNAG antibody titers were assessed by ELISA on days 0 and 35 post-immunization. PNAG antibody functional activity was assessed by complement component C1q deposition, bactericidal or opsonophagocytic killing assays. Heifer estrus cycles were synchronized using three doses of 25 mg of dinoprost tromethamine (EstrumateTM) at 14-day intervals, prior to insemination with fertile frozen/thawed semen and inoculation with 106 T. foetus organisms into the anterior vagina beneath the external cervical os, followed by100 mcg gonadorelin (FacrelTM). Culture and pregnancy testing were performed in a blinded fashion. Vaginal mucus was evaluated weekly for infection status from day 4 until day 32 post insemination. The mucus was immediately inoculated into an InpouchTM container and cultured at 37oC. On day 32 post-insemination pregnancy was evaluated by trans-rectal ultrasonography. From day 32 until 4 months post-insemination, pregnancy evaluations were repeated and vaginal cultures collected at monthly intervals. T. foetus was identified by morphology and motility on days 1 to 4 and day 7. Fisher’s Exact Test compared proportions of infected and non-infected animals in the control and vaccine groups.
PNAG-specific antibody titers were achieved after vaccination. 15 /16 heifers remained vaginal culture positive until day 32 post-insemination. On day 60, 14/16 heifers were culture positive and on day 90, 5/16 heifers were culture positive. At the study conclusion (day 120), 3/16 animals were culture positive, including one control pregnant cow and two vaccinated non-pregnant cows. The rates of positive culture were not significantly different between vaccinates and controls (P > 0.1). At day 32 post-insemination, 3 control heifers and 2 vaccinated cows were pregnant. One vaccinated cow aborted before 4 months of gestation leaving 3 control pregnant heifers and 1 vaccinated pregnant heifer at the study end. Pregnancy rates were not significantly different between groups (P > 0.1). Further in vitro assays determined antibodies elicited were also non-protective in bacterial killing assays.
The study illustrates considerable potential for the development of a novel vaccine for T. foetus. First, unlike bulls, cows do clear the infection and, presumably, the immune system plays an important role. With this study’s experimental challenge, only 1/16 cows appeared to clear the (vaginal) infection before embryonic signaling (approximately day 18) and before placental attachment (approximately day 32). The challenge dose of 106 organisms may be a factor and further studies should include a vaccination trial using natural insemination by infected bulls. The current study also showed that vaginal infection is often tolerated during pregnancy, but abortion did not occur. In contrast to PNAG and Specol vaccination of pigs and horses, that elicited functional and protective antibodies against other pathogens, in cows this same formulation elicited non-protective and non-functional antibodies.