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RB 51 Vaccine USDA, APHIS, VS and F. M. Enright Louisiana State University
This afternoon I will provide you all with a brief history of RB51 Brucella Vaccine. As I speak, a number of field studies testing the safety of this vaccine are underway in at least seven states.
As you have just learned in the previous presentations, Brucellosis in cattle in the United States is becoming a rare disease. Considerable effort and funds are being expended to find the few remaining infected cattle. Until these cattle are found, selected populations of Brucella-free cattle must be protected. I’m sure you may be asking - "so what is wrong with continuing to use Stain-19 vaccine to protect these at risk cattle?" Strain-19 vaccine is a good vaccine, and it has served us well as a valuable tool in controlling the spread of Brucellosis in cattle. Strain-19 vaccine however, has one particularly annoying characteristic. Too often Strain-19 vaccination causes some vaccinated cattle to develop prolonged positive serology to the standard Brucellosis diagnostic tests. Because we cannot easily distinguish between a positive test due to vaccination and one due to real infection, we spend considerable time and money making sure that "positive" animals really are not infected. Every positive cow must be considered infected until proven otherwise. Therefore for every vaccination titer we chase, we may miss that one last field infection.
Scientists conducting Brucella research for the last 40 years have been looking for a vaccine which is as effective as Strain-19 but which will not cause positive postvaccinal serological reactions.
Here I have a picture of a premature dead calf from an experimentally infected cow. This slide should remind us all that Brucellosis really is a disease which can be devastating to livestock production.
We were guided in our research for a vaccine to replace Strain-19 by looking for a candidate vaccine which is safe and as effective as Strain-19, which results in long lasting immunity, and one that does not result in serological reactions that interfere with diagnosis. I might add that Strain-19 vaccine itself cannot meet all of these criteria - and as you will see neither does RB51.
No matter which vaccine is chosen to control Brucellosis, it was recognized early that in order to be effective the vaccine strain or antigen must persist in the lymphoid tissues of the host long enough to stimulate the right type of immunity. Killed vaccines are eliminated from the host before the immune system gets a good look at them. Therefore, killed Brucella vaccines are generally not very effective in protecting cattle against infection.
In the early ‘80’s, Dr. Schurig at Virginia Tech discovered a mutant of smooth, virulent B. abortus strain 2308 which demonstrated rough cultural characteristics. He demonstrated that this mutant named RB51 could not synthesize the O-side chain of B. abortus lipopolysaccharide. He initially demonstrated that RB51 was rapidly cleared from the tissues of experimentally infected mice. In fact, RB51 was cleared so rapidly that he was concerned that it may prove to be an unacceptable vaccine candidate because it was too attenuated. Fortunately mice are not cows or goats for we were able to demonstrate that the organism was not cleared as rapidly from the lymph nodes of RB51-inoculated goats or cattle. Another important characteristic of RB51 was that, after passage through mice, goats, and cattle, the mutant remained rough and did not revert back to the smooth, virulent characteristics of its parent strain. Even more important, none of the animals experimentally inoculated with RB51 developed antibodies to the O-side chain. This meant that their sera would not test positive to various Brucellosis diagnostic tests because these tests are based on the detection of antibodies to the O-side chain of B. abortus.
These early experiments also showed that RB51 under certain conditions could infect cattle, persist in their tissues, and infect their pregnant uterus. One cow was infected and delivered a term, infected calf. The RB51 isolated from this cow and calf was, however, still attenuated and rough. It is important to remember that pregnant cattle inoculated with Strain-19 may abort also.
By the late 1980’s, we were testing RB51 for its ability to immunize mice and demonstrated that the immunity in mice was due to T-lymphocytes and did not depend solely on antibodies. Experiments in the laboratory also demonstrated that RB51 would infect and grow within trophoblastic cells of the cow’s placenta but failed to cause significant toxic injury to these cells. These experiments predicted that even though RB51 could infect the pregnant uterus, the bacteria would be unlikely to cause abortions. This prediction was confirmed by studies in which late gestational pregnant goats were infected with RB51. In these studies the RB51 was directly inoculated into the pregnant uterus. The fetal goats and their placental tissues were infected with RB51, but they failed to abort and were delivered alive at term. The molecular mechanisms responsible for fetal and placental infection without an abortion are not yet understood.
Taken together, all of the preliminary data had convinced Drs. Schurig and Enright that RB51 might be a suitable vaccine candidate to replace Strain-10. RB51 was stable; it persisted long enough in lymph nodes to stimulate immunity; it was relatively non-pathogenic in comparison to Strain-19; and it did not result in the production of antibodies which react in the standard diagnostic tests for Brucellosis.
In case you are wondering what is meant by rough or smooth, this slide is a diagram of the surface of smooth Brucella organisms. You will note there are numerous chains with square links extending from the surface of the bacterial wall. Those chains represent the O-side chain of the Brucella lipopolysaccharide. Infected cattle and some Strain-19 vaccinated cattle make antibodies to these square links. It is these antibodies which we detect in the Brucellosis diagnostic tests. In contrast, the surface of RB51 does not have the square links, and the animals exposed to RB51 do not make antibodies to them.
This is a histological section of a superficial cervical lymph node biopsied from a cow which had been inoculated seven days earlier with RB51. The large pale oval structure in the lymph node is a follicle of lymphocytes which indicates that this cow is mounting an immune response to RB51. The three small round pink structures on the left side of the slide are nests of phagocytic white blood cells containing RB51. By culturing this lymph node, we can recover the RB51 and determine how long the bacteria persists in this tissue following vaccination.
The next series of slides will show the results of the first preliminary studies in cattle to prove that RB51 can protect cattle as well as Strain-19. This data was first presented in March of 1990. The objective of the study was to demonstrate RB51 protection against experimental challenge. In this study groups of adult cattle were vaccinated with either Strain-19, RB51, or given saline as a control. The cattle were all bred and then challenged with virulent B. abortus strain 2308 while in the late stages of pregnancy.
Following challenge as each cow gave birth or aborted, we cultured both the cow and the calf or aborted fetus to recover the challenge strain of B. abortus. Any cow was considered infected if either she or her calf or fetus were culture positive.
The results indicate that nearly all of the saline control cattle were infected. Infection rates were also relatively high in the Strain-19 vaccinated cattle while only two of the seven cows exposed to RB51 were infected.
This study suggested that RB51 protected these cattle against infection. Subsequent studies have also demonstrated that cattle orally vaccinated with RB51 are protected against infection when challenged with virulent B. abortus. In this study only two of ten vaccinated cattle were infected while eight of ten of the control cattle were infected.
These preliminary studies were very encouraging and resulted in additional studies using larger numbers of cattle. These studies were conducted by Dr. Norm Cheville’s research group at the National Animal Disease Center in Ames, Iowa. Many of these studies are still ongoing. The key findings of these experiments will be summarized in the following overheads.
A large number of calves of different ages varying from 6 weeks old to ten months old were inoculated with RB51. At various intervals following inoculation, the draining lymph nodes in these calves were biopsied. RB51 was shown to persist in these lymph nodes for up to 12 weeks in the cattle vaccinated at ten months of age. Further, none of RB51 recovered from these calves had reverted. The RB51 had remained stable and had caused no reactions to only slight reactions in the lymphoid tissues of these cattle. Most importantly, the calves did not make anti-O-side chain antibodies and remained negative to the Brucella diagnostic tests.
Next, Dr. Cheveille’s group challenged cows at 5 to 6 months of gestation with virulent B. abortus. These cows had been vaccinated as calves at 3, 5, 7, and 10 months of age with RB51. The RB51 vaccinates demonstrated protection against both infection and abortion when compared to the saline control cattle. The level of protection in these RB51 vaccinated cattle is similar to the level of protection observed in calves vaccinated with Strain-19.
In other biosafety related studies, the research group at NADC and USDA/APHIS/VS veterinarians reexamined the risk of using RB51 in pregnant cattle. These studies were conducted in two ways. First, over 350 cattle at different stages of gestation were inoculated subcutaneously with one billion viable RB51 bacteria. These animals were monitored postvaccination and selected animals were cultured post-delivery. To date, none have aborted due to infection with RB51.
In the second series of experiments, ten cows in the sixth month of gestation were inoculated intravenously with 10 billion viable RB51. This does of bacteria is ten times that normally used. Five cows were sacrificed 8 weeks postinoculation and five were allowed to continue their pregnancies. Eighty percent of these cattle demonstrated placental infections with RB51 but none aborted. One cow gave birth to a live, weak, premature RB51 infected calf. In contrast with these results, experimental intravenous inoculation of pregnant cattle with Strain-19 typically results in placentitis and abortions at 16 to 42 days postinoculation. In one study Strain-19 caused abortions in 100% of the inoculated cows. These studies thus support the preliminary data obtained in pregnant goats and in vitro culture systems that RB51 is less virulent and abortogenic than Strain-19.
As mentioned in the beginning of this presentation, approximately 723 cattle in selected herds in seven states have been exposed subcutaneously to one billion RB51 and today none have been observed to have immediate or delayed reactions including no abortions in those pregnant at the time of exposure to RB51.
In conclusion, twelve years of research on RB51 have demonstrated that the "vaccine" is less virulent than Strain-19 and provides cattle with levels of protection equal to that observed with Strain-19, and RB51 vaccinated cattle do not develop positive reactions with the commonly used Brucellosis diagnostic tests.
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