Indirect enzyme-linked immunosorbent assay for detection of Brucella melitensis-specific antibodies in goat milk.

Brucella melitensis is the cause of brucellosis in sheep and goats, which often results in abortion. Few cases of B. melitensis infection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection of B. melitensis-specific antibodies in goat milk. Brucella salt-extractable protein extract was employed as an antigen, and a horseradish peroxidase-labeled polyclonal anti-goat antibody was used as an anti-species conjugate. Thirteen of 13 (100%) individual infected goat milk samples tested positive and 134 of 134 (100%) uninfected bulk milk samples tested negative by the developed iELISA. Three positive milk samples with high, medium, and low absorbance values were used to simulate one positive animal in an otherwise negative herd. By this estimation, one high-titer animal could be detected in a herd of >1,600 animals. Detection estimates for medium- and low-titer animals were one positive animal per herd of <200 and 50 animals, respectively. Based on this estimation, it is recommended that herds be sampled in groups of 50 animals or less for bulk milk testing. The iELISA developed for this study was found to be sensitive and specific and shows potential for use as a bulk milk test for the detection of B. melitensis-specific antibodies in goat milk.

Characterization of the caprine model for ruminant brucellosis.

The relationship between man, the goat, and brucellosis is historical. Today Brucella melitensis and Brucella abortus pose a serious economic and public health threat in many countries throughout the world. Infection of pregnant goats and sheep with B. melitensis results in abortion during the third trimester of pregnancy. Although nearly eradicated in the US, bovine brucellosis is still a problem in many countries and the potential for re-infection of domestic stock from wildlife reservoirs in this country is a regulatory nightmare. Humans infected with this pathogen develop undulant fever, which is characterized by pyrexia, arthritis, osteomyelitis, and spondylitis. Although available for both organisms, currently available vaccines have problems ranging from false positive serological reactions to limited efficacy in different animal species. With the continued need for new and better vaccines, we have further developed a goat model system to test new genetically derived strains of B. melitensis and B. abortus for virulence as measured by colonization of maternal and fetal tissues, vaccine safety, and vaccine efficacy.

Major histocompatibility complex class I and II expression on macrophages containing a virulent strain of Brucella abortus measured using green fluorescent protein-expressing brucellae and flow cytometry.

Immune responses appropriate for control of an intracellular pathogen are generated in mice infected with Brucella abortus, shown by the ability of T cells to adoptively transfer resistance to naive mice. The infection nevertheless persists for months. It was hypothesized that one factor in maintaining the infection despite the presence of immune T cells was suboptimal expression of major histocompatibility complex (MHC) molecules on macrophages containing brucellae. This would allow B. abortus to elude detection by the host's immune system. To test this, B. abortus organisms expressing green fluorescent protein (GFP-Brucella) were constructed and three-color flow cytometry used to evaluate MHC expression on macrophages following in vitro or in vivo infection. When infected in vitro, the levels of MHC class I and class II expression on J774 macrophages containing GFP-Brucella were the same or higher than on macrophages without GFP-Brucella in the same cultures. Similarly, the MHC expression was higher on GFP(+) peritoneal exudate cells following infection or phagocytosis of heat-killed GFP-Brucella than it was on uninfected peritoneal exudate cells. Following in vivo infection of mice the level of MHC class I and II expression on GFP(+) cells in their spleens (the main site of infection) also tended to be as high as or higher than that on the GFP-negative cells. The only in vivo GFP(+) cells that showed a decreased MHC expression was a population of splenic Mac1(+) cells recovered from interferon-gamma gene-disrupted mice at the time of their death due to an overwhelming number of bacteria per spleen. Overall, it was concluded that decreased MHC expression is not a general principle associated with brucella infection of macrophages and thus not likely to contribute to maintenance of the chronic infection.

Pathogenicity and protective activity in pregnant goats of a Brucella melitensis Deltaomp25 deletion mutant.

The Brucella melitensis mutant BM 25, which lacks the major 25 kDa outer membrane protein Omp25, has previously been found to be attenuated in the murine brucellosis model. In the present study, the capacity of the Deltaomp25 mutant to colonise and cause abortions in the caprine host was evaluated. The vaccine potential of BM 25 was also investigated in goats. Inoculation of nine pregnant goats in late gestation with the B. melitensis mutant resulted in 0/9 abortions, while the virulent parental strain, B. melitensis 16M, induced 6/6 dams to abort (P<0.001, n=6). BM 25 also colonised fewer adults (P<0.05, n=6) and kids (P<0.01, n=6) than strain 16M. The Deltaomp25 mutant was found capable of transient in vivo colonisation of non-pregnant goats for two weeks post-infection. Owing to the ability of BM 25 to colonise both non-pregnant and pregnant adults without inducing abortions, a vaccine efficacy study was performed. Vaccination of goats prior to breeding with either BM 25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in 100 per cent protection against abortion following challenge in late gestation with virulent strain 16M (P<0.05, n=7). However, unlike strain Rev. 1, BM 25 does not appear to cause abortions in late gestation based on this study with a small number of animals. The B. melitensis Deltaomp25 mutant, BM 25, may be a safe and efficacious alternative to strain Rev. 1 when dealing with goat herds of mixed age and pregnancy status.

Attenuation of a Brucella abortus mutant lacking a major 25 kDa outer membrane protein in cattle.

OBJECTIVE: To determine the virulence of a Brucella abortus mutant, BA25, lacking a major 25 kd outer membrane protein (Omp25) in cattle. ANIMALS: 20 mixed-breed heifers in late gestation. PROCEDURE: 10 heifers were inoculated with 1 x 10(7) colony-forming units of the Omp25 mutant via the conjunctival sac, and an equal number were infected with the virulent parental strain B. abortus 2308. The delivery status of the dams was recorded, and colonization was assessed following necropsy. The ability of BA25 to replicate inside bovine phagocytes and chorionic trophoblasts was also evaluated in vitro because of the propensity of virulent brucellae to replicate inside these cells in vivo. RESULTS: The parental strain induced abortions in 5 of 10 inoculated cattle, whereas only 1 of 10 dams exposed to BA25 aborted. Brucella abortus strain 2308 colonized all of the cow-calf pairs and induced Brucella-specific antibodies in 100% of the dams. In contrast, BA25 was isolated by bacteriologic cultural technique from 30% of the calves and 50% of the inoculated dams (n = 10). Of the 10 heifers inoculated with BA25, 4 did not develop Brucella-specific antibodies nor were they colonized by the mutant strain. In bovine macrophages and chorionic trophoblasts, BA25 replicated in significantly lower numbers than the virulent parental strain (n = 3). CONCLUSIONS AND CLINICAL RELEVANCE: The 25 kd outer membrane protein may be an important virulence factor for B. abortus in cattle. The attenuation of the Omp25 mutant in cattle may involve the inability of BA25 to replicate efficiently in bovine phagocytes and chorionic trophoblasts.

Oral vaccination of sexually mature pigs with Brucella abortus vaccine strain RB51.

OBJECTIVE: To develop a novel oral vaccine delivery system for swine, using the rough vaccine strain of Brucella abortus. ANIMALS: 56 crossbred pigs from a brucellosis-free facility. PROCEDURE: In 3 separate experiments, pigs were orally vaccinated with doses of 1 x 10(9) to > 1 x 10(11) CFU of strain RB51 vaccine. The vaccine was placed directly on the normal corn ration, placed inside a whole pecan, or mixed with cracked pecans and corn. RESULTS: Oral vaccination of pigs with vaccine strain RB51 resulted in a humoral immune response to strain RB51 and short-term colonization of the regional lymph nodes. CONCLUSIONS AND CLINICAL RELEVANCE: A viscous liquid such as Karo corn syrup in association with pecans that scarify the oral mucosa are necessary when placing the live vaccine directly onto corn or other food rations. Doses of > 1 x 10(11) CFU of RB51 organisms/pig in this mixture ensures 100% colonization of regional lymph nodes via the oral route. This method may allow an efficient and economical means to vaccinate feral swine for brucellosis.

Re-examination of the role of the Brucella melitensis HtrA stress response protease in virulence in pregnant goats.

Based on previously reported studies describing the experimental infection of pregnant goats with B. melitensis strain RWP5, we proposed that the HtrA protease plays an important role in the virulence of B. melitensis in its natural ruminant host. Subsequent studies, however, have shown that RWP5 is actually an htrA cycL double mutant. In order to definitively evaluate the role of the B. melitensis htrA in virulence, we constructed an authentic htrA mutant and examined this strain in pregnant goats. The findings of these studies indicate that the contribution of the htrA gene product to the virulence of B. melitensis in its natural host is not as great as was previously proposed.

Attenuation and immunogenicity of a Brucella abortus htrA cycL double mutant in cattle.

PHE1 is a htrA cycL double gene deletion mutant of virulent Brucella abortus strain 2308 (S2308) which has previously been evaluated in the murine and caprine models of bovine brucellosis. This report describes the results of studies conducted with this mutant in the natural bovine host. Six sexually mature, non-gravid heifers were inoculated via the conjunctival sac with 1 x 10(10) colony forming units (CFU) of either the parental S2308 or the htrA cycL gene deletion mutant, PHE1. At 4, 7 and 11 days post-inoculation, PHE1 was found to colonize the bovine host at lower levels than S2308. In a second experiment, eight heifers in mid-gestation were infected with 1 x 10(7) CFU of either strain via the conjunctival sac. The virulent S2308 caused abortions or weak calves in 4/4 cows, while all four cows infected with PHE1 had healthy calves. Furthermore, PHE1 exhibited decreased resistance to killing by cultured bovine neutrophils and macrophages compared to the parental strain. These studies demonstrate that the B. abortus htrA cycL gene deletion mutant PHE1 is highly attenuated in the bovine host when compared to the virulent parental S2308.

A multiplex approach to molecular detection of Brucella abortus and/or Mycobacterium bovis infection in cattle.

A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a "gold standard," was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortus shedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of B. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.

Biosafety of Brucella abortus strain RB51 for vaccination of mature bulls and pregnant heifers.

OBJECTIVE: To determine shedding and colonization profiles in mature sexually intact bulls and pregnant heifers after vaccination with a standard calfhood dose of Brucella abortus strain RB51 (SRB51). ANIMALS: 6 sexually mature 3-year-old Jersey bulls and 7 mixed-breed heifers in midgestation. PROCEDURE: Bulls and pregnant heifers were vaccinated IM with the standard calfhood dose of 3x10(10) colony-forming units of SRB51. After vaccination, selected body fluids were monitored weekly for vaccine organism shedding. Pathogenesis was monitored in bulls by weekly breeding soundness examination and, in heifers, by delivery status of the calf. Vaccine organism colonization was assessed by obtaining select tissues at necropsy for bacterial culture. Serologic analysis was performed by use of numerous tests, including complement fixation, an SRB51-based ELISA, and immunoblot analysis. RESULTS: After vaccination, none of the vaccinated bulls or heifers shed SRB51 in their secretions. Results of breeding soundness examination for bulls were normal as was delivery status of the pregnant heifers (6 live births, 1 dystocia). At necropsy, SRB51 was not recovered from any of the selected tissues obtained from bulls, heifers, or calves; however, serologic analysis did detect SRB51-specific antibodies in all cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination with the standard calfhood dose of SRB51 administered IM was not associated with shedding or colonization in sexually mature bulls or pregnant heifers. Also, under conditions of this study with small numbers of animals, IM vaccination with SRB51 does not appear to cause any reproductive problems when administered to sexually mature cattle.

Protection against infection and abortion induced by virulent challenge exposure after oral vaccination of cattle with Brucella abortus strain RB51.

OBJECTIVES: To determine efficacy of orally administered Brucella abortus vaccine strain RB51 against virulent B abortus challenge exposure in cattle as a model for vaccination of wild ungulates. ANIMALS: 20 mixed-breed beef cattle obtained from a brucellosis-free herd. PROCEDURE: Sexually mature, Brucella-negative beef heifers were vaccinated by mixing > 10' viable RB51 organisms or diluent with their feed. Heifers were fed individually and consumed their entire ration. Each heifer received approximately 3 X 10' colony-forming units (CFU). Six weeks after oral vaccination, heifers were pasture-bred to brucellosis-free bulls. At approximately 186 days' gestation, heifers were challenge exposed conjunctively with 107 CFU of virulent B abortus strain 2308. RESULTS: Vaccination with the rough variant of B abortus RB51 did not stimulate antibodies against the O-polysaccharide (OPS) of B abortus. After challenge exposure and parturition, strain 2308 was recovered from 80% of controls and only 20% of vaccinates. Only 30% of the vaccinates delivered dead, premature, or weak calves, whereas 70% of the controls had dead or weak calves. CONCLUSIONS: Cattle vaccinated orally with the rough variant of B abortus strain RB51 develop significant (P < 0.05) protection against abortion and colonization and do not produce OPS-specific antibodies. Clinical Relevance-Results encourage further investigation into use of strain RB51 to vaccinate wild ungulates (elk and bison) orally.

Safety of Brucella abortus strain RB51 in Bison.

To determine the safety of Brucella abortus strain RB51 (SRB51) vaccine in American bison (Bison bison), 31 animals from a herd with brucellosis were used. In October 1996, 10 adult bison males and seven calves were vaccinated with the standard calfhood cattle dose of 1.8 x 10(10) colony forming units (CFU) of SRB51 subcutaneously while the adult females received the standard adult cattle dose of 1 x 10(9) CFU. Western immunoblot indicated the presence of SRB51 antibodies following vaccination. To evaluate prolonged bacterial colonization of tissues, the adult males, calves, and three adult females were divided into two groups which were slaughtered at either 13 or 16 wk post-vaccination. At necropsy, tissue samples were obtained for B. abortus culture from the liver, spleen, lymph nodes, and reproductive tract of each animal. While B. abortus field strain was cultured from one adult bull, no SRB51 was isolated from any of the animals. Seven pregnant females were monitored until parturition for signs of abortions and fetal lesions. Six cows delivered healthy calves and one delivered a dead full-term calf that was brucellae negative. Based on these results, administration of SRB51 to bison did not cause prolonged bacterial colonization of tissues in calves, adult males, or adult females. Furthermore, SRB51 did not induce abortions following vaccination in the second month of gestation.

A Brucella melitensis high-temperature-requirement A (htrA) deletion mutant is attenuated in goats and protects against abortion.

It has been previously demonstrated that a Brucella melitensis high-temperature-requirement A (htrA) deletion mutant is more susceptible to oxidative killing in vitro than the parental strain and is attenuated in mice. To evaluate the contribution of the B melitensis HtrA protease to virulence in ruminants, the capacity of the B melitensis htrA mutant RWP5 to produce abortion in goats was compared to that of the virulent parental strain 16M. Experimental infection with strain 16M caused abortion in eight of 12 pregnant nannies, while none of the 12 nannies inoculated with RWP5 aborted. Furthermore, intramuscular injection of fetuses in utero with RWP5 led to colonisation of the fetus with subsequent colonisation of the nanny, but no abortion was observed. Nannies vaccinated with RWP5 showed complete protection against abortion when challenged with 16M during the third trimester of pregnancy. However, these animals were not protected from colonisation by 16M. The results presented here clearly indicate that the B melitensis htrA gene product contributes to pathogenesis in goats, but the utility of B melitensis htrA mutants as vaccines in this host appears to be limited.

Stability of equine infectious anemia virus in Aedes aegypti (Diptera: Culicidae), Stomoxys calcitrans (Diptera:Muscidae), and Tabanus fuscicostatus (Diptera:Tabanidae) stored at -70 degrees C.

Equine infectious anemia virus (EIAV) was injected intrathoracically into Aedes aegypti, Stomoxys calcitrans, and Tabanus fuscicostatus, and fed to Ae. aegypti in suspensions of either artificial blood of Eagle's Minimum Essential Medium. Insects were stored at -70 degrees C for up to 9 months before testing for the presence of EIAV. The viral tissue culture titers detected from stored insects were similar to those from insects tested at time 0.

Behaviour of a high-temperature-requirement A (HtrA) deletion mutant of Brucella abortus in goats.

Previous studies have shown that high-temperature-requirement A (HtrA) mutants of Brucella abortus are more sensitive to oxidative killing in vitro, are less able to survive in cultured murine macrophages and are attenuated in BALB/c mice. To measure the effect of an HtrA mutation on the virulence of B abortus in ruminants, pregnant goats in late gestation were exposed to infection by the conjunctival route with B abortus 2308 or an isogenic htrA mutant, PHE1. Infection with either 2308 or PHE1 resulted in abortion, but the serological responses to infection were consistent with 2308 but variable with PHE1. Strain 2308 was recovered post mortem both from aborted fetuses and infected dams, whereas PHE1 was recovered from neither. Nevertheless, short term studies revealed that PHE1 could be recovered from infected goats for up to two weeks after infection, suggesting that although the HtrA mutation may change the colonising ability of B abortus, the virulence of the mutant in pregnant goats is not reduced.

Efficacy of inactivated whole-virus and subunit vaccines in preventing infection and disease caused by equine infectious anemia virus.

We report here on a series of vaccine trials to evaluate the effectiveness of an inactivated equine infectious anemia virus (EIAV) whole-virus vaccine and of a subunit vaccine enriched in EIAV envelope glycoproteins. The inactivated vaccine protected 14 of 15 immunized ponies from infection after challenge with at least 10(5) 50% tissue culture-infective doses of the homologous prototype strain of EIAV. In contrast, it failed to prevent infection in any of 15 immunized ponies that were challenged with the heterologous PV strain. Levels of PV virus replication and the development of disease, however, were significantly reduced in 12 of the 15 ponies so challenged. The subunit vaccine prevented infection from homologous challenge in four of four ponies tested but failed to prevent infection in all four challenged with the PV strain. Two of the four subunit vaccinates had more severe symptoms of equine infectious anemia than nonimmunized ponies infected in parallel. Both vaccines stimulated EIAV-specific cell-mediated immunity. The in vitro lymphoproliferative response was shown to be mediated by T lymphocytes and appeared to be indistinguishable from that induced by EIAV infection. Significant differences were observed in the in vivo lymphocyte responses following challenge with the two virus strains. While peripheral blood mononuclear cells from the inactivated virus vaccinates were equally stimulated by both the prototype and PV strains, the subunit vaccinates challenged with PV exhibited lower levels of spontaneous proliferation and serine esterase activity. This diminished cellular response to PV was correlated with more severe clinical disease in the same ponies. These studies demonstrate for the first time that both an EIAV inactivated whole-virus vaccine and a viral envelope glycoprotein-based subunit vaccine can provide protection against rigorous challenge levels of homologous virus but are unable to protect against similar challenge levels of a heterologous virus. Moreover, the data demonstrate that protection can be achieved in the absence of detectable levels of virus-specific neutralizing antibody in the vaccine recipients at the time of virus challenge. While vaccine-induced virus-specific cell-mediated immune responses were detected, their role in conferring protection was not obvious. Nevertheless, protection from disease appeared to be correlated with the induction of high levels of serine esterase activity following challenge. A significant observation is that while the whole-virus vaccine was usually capable of preventing or markedly moderating disease in the PV-infected ponies, the subunit vaccine appeared to have a high potential to enhance the disease induced by PV infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Equine infectious anemia: prospects for control.

Equine infectious anemia has been managed in most countries by the imposition of testing and quarantine regulations. In the United States, about 700,000 of the more than 7,000,000 horses are tested annually. As long as the status of greater than 90% of the horse population remains unknown and horses are transported and congregate in a relatively unrestricted manner, EIA will continue to exact its toll. Therefore, it is incumbent on the scientific community to continue to develop and refine practical and sensitive diagnostic tests for EIA which will be used in an expanding market, to reduce the number of untested horses and to increase the accuracy of test results. Under ideal conditions, EIA can spread rapidly in a localized population with potentially devastating results. Although strict adherence to sanitary regulations will minimize the likelihood of epizootics, the existence of a large reservoir of untested horses with occasional contact with uninfected test-negative horses will ensure the continued transmission of EIAV. The change of this transmission occurring as a result of human intervention can be eliminated but it is not possible to eliminate the threat posed by blood feeding insects. If these "chance encounters" between an untested EIAV infected horse and a test-negative horse occur under field conditions where horse flies are abundant and the proximate distance between the horses is minimal, transmission is efficient if the quantity of EIAV in the blood of the donor horse is high.(ABSTRACT TRUNCATED AT 250 WORDS)