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ABSTRACTStrain superinfection affects the dynamics of epidemiological spread of pathogens through a host population. Superinfection has recently been shown to occur for two genetically distinct strains of the tick-borne pathogen Anaplasma marginalethat encode distinctly different surface protein variants. Superinfected animals could serve as a reservoir for onward transmission of both strains if the tick vector is capable of acquiring and transmitting both strains. Whether competition among strains during development within the tick vector, which requires sequential invasion and replication events, limits colonization and subsequent transmission to a single strain is unknown. We tested this possibility by acquisition feeding Dermacentor andersoniticks on a reservoir host superinfected with the genetically distinct St. Maries and EMΦ strains. Although the St. Maries strain consistently maintained higher bacteremia levels in the mammalian host and the EMΦ strain had an early advantage in colonization of the tick salivary glands, individual ticks were coinfected, and there was successful transmission of both strains. These results indicate that a genetically distinct A. marginalestrain capable of superinfecting the mammalian host can subsequently be cotransmitted and become established within the host population despite the presence of an existing established strain.

Strain superinfection affects the dynamics of epidemiological spread of pathogens through a host population. Superinfection has recently been shown to occur for two genetically distinct strains of the tick-borne pathogen Anaplasma marginale that encode distinctly different surface protein variants. Superinfected animals could serve as a reservoir for onward transmission of both strains if the tick vector is capable of acquiring and transmitting both strains. Whether competition among strains during development within the tick vector, which requires sequential invasion and replication events, limits colonization and subsequent transmission to a single strain is unknown. We tested this possibility by acquisition feeding Dermacentor andersoni ticks on a reservoir host superinfected with the genetically distinct St. Maries and EM strains. Although the St. Maries strain consistently maintained higher bacteremia levels in the mammalian host and the EM strain had an early advantage in colonization of the tick salivary glands, individual ticks were coinfected, and there was successful transmission of both strains. These results indicate that a genetically distinct A. marginale strain capable of superinfecting the mammalian host can subsequently be cotransmitted and become established within the host population despite the presence of an existing established strain.

ABSTRACTBronchopneumonia is a population-limiting disease in bighorn sheep in much of western North America. Previous investigators have isolated diverse bacteria from the lungs of affected sheep, but no single bacterial species is consistently present, even within single epizootics. We obtained high-quality diagnostic specimens from nine pneumonic bighorn sheep in three populations and analyzed the bacterial populations present in bronchoalveolar lavage specimens of seven by using a culture-independent method (16S rRNA gene amplification and clone library analyses). Mycoplasma ovipneumoniaewas detected as a predominant member of the pneumonic lung flora in lambs with early lesions of bronchopneumonia. Specific PCR tests then revealed the consistent presence of M. ovipneumoniaein the lungs of pneumonic bighorn sheep in this study, and M. ovipneumoniaewas isolated from lung specimens of five of the animals. Retrospective application of M. ovipneumoniaePCR to DNA extracted from archived formalin-fixed, paraffin-embedded lung tissues of historical adult bighorn sheep necropsy specimens supported the association of this agent with bronchopneumonia (16/34 pneumonic versus 0/17 nonpneumonic sheep were PCR positive [P< 0.001]). Similarly, a very strong association was observed between the presence of one or more M. ovipneumoniaeantibody-positive animals and the occurrence of current or recent historical bronchopneumonia problems (seropositive animals detected in 9/9 versus 0/9 pneumonic and nonpneumonic populations, respectively [P< 0.001]). M. ovipneumoniaeis strongly associated with bronchopneumonia in free-ranging bighorn sheep and is a candidate primary etiologic agent for this disease.

ABSTRACTBabesia bovisis a deadly disease of cattle resulting in severe economic losses in the vast regions of the world where it is endemic. If reintroduced into the United States, babesiosis would cause significant mortality in the nai¨ve cattle population. In order to address the risk to U.S. cattle, it is essential to quantify the transovarial transmission efficiency in adult female Boophilus microplusticks following acquisition feeding on persistently infected cattle. This study tested the hypothesis that infection rates are the same for larval progeny derived from females fed to repletion during persistent or acute infection. Increasing parasite levels during acute infection correlated with an increasing number of females harboring kinetes detectable in hemolymph (r= 0.9). The percent infected larvae ranged from 0 to 20% when derived from females fed to repletion on persistently infected calves and from 4 to 6% when derived from females fed to repletion during acute parasitemia. There was no significant difference in infection rates of larval progeny, implying that the risk associated with the introduction of either persistently infected or acutely infected cattle is equal. Parasite levels ranged from 2.4 × 102to 1.9 × 105in 3-day-fed larvae derived from females fed to repletion on persistently infected cattle. One group of larvae failed to transmit the parasite, suggesting that a threshold level of parasites must be obtained by larval progeny via transovarial transmission in order for larvae to deliver sufficient parasites to infect a nai¨ve host.

ABSTRACTAnaplasma marginale, a rickettsial pathogen, evades clearance in the animal host by antigenic variation. Under immune selection, A. marginaleexpresses complex major surface protein 2 mosaics, derived from multiple donor sequences. However, these mosaics have a selective advantage only in the presence of adaptive immunity and are rapidly replaced by simple variants following transmission.

ABSTRACTThe protozoan parasite Babesia bovis, a reemerging threat to U.S. cattle, is acquired by adult female ticks of the subgenus Boophilusand is transovarially transmitted as the kinete stage to developing larval offspring. Sporozoites develop within larvae and are transmitted during larval feeding on a bovine host. This study evaluated the efficiency of B. bovisinfection within Rhipicephalus(Boophilus) microplusfollowing acquisition feeding on acutely parasitemic cattle. Parasite levels were quantified in blood from experimentally infected cattle and within hemolymph and larvae derived from acquisition-fed female B. microplus. There was a positive correlation between blood parasite levels in acutely parasitemic cattle and kinete levels in the hemolymph of adult female Boophilusticks following acquisition feeding; however, there was no relationship between kinete levels in females and infection rates of larval progeny. Boophilus microplusfemales that acquisition fed produced larval progeny with infection rates of 12% to 48%. Importantly, larvae derived from replete females with very low levels of kinete infection, as demonstrated by microscopy and PCR, had infection rates of 22% to 30% and transmitted B. bovisduring transmission feeding. These data demonstrate that although hemolymph infection may be undetectable, transmission to larval progeny occurs at a level which ensures transmission to the bovine host.

Bacterial pathogens in the genera Anaplasma and Ehrlichia encode a protein superfamily, pfam01617, which includes the predominant outer membrane proteins (OMPs) of each species, major surface protein 2 (MSP2) and MSP3 of Anaplasma marginale and Anaplasma ovis, Anaplasma phagocytophilum MSP2 (p44), Ehrlichia chaffeensis p28-OMP, Ehrlichia canis p30, and Ehrlichia ruminantium MAP1, and has been shown to be involved in both antigenic variation within the mammalian host and differential expression between the mammalian and arthropod hosts. Recently, complete sequencing of the A. marginale genome has identified an expanded set of genes, designated omp1-14, encoding new members of this superfamily. Transcriptional analysis indicated that, with the exception of the three smallest open reading frames, omp2, omp3, and omp6, these superfamily genes are transcribed in A. marginale-infected erythrocytes, tick midgut and salivary glands, and the IDE8 tick cell line. OMPs 1, 4, 7 to 9, and 11 were confirmed to be expressed as proteins by A. marginale within infected erythrocytes, with expression being either markedly lower (OMPs 1, 4, and 7 to 9) or absent (OMP11) in infected tick cells, which reflected regulation at the transcript level. Although the pfam01617 superfamily includes the antigenically variable MSP2 and MSP3 surface proteins, analysis of the omp1-14 sequences throughout a cycle of acute and persistent infection in the mammalian host and tick transmission reveals a high degree of conservation, an observation supported by sequence comparisons between the St. Maries strain and Florida strain genomes.

ABSTRACTBacterial pathogens in the genera Anaplasmaand Ehrlichiaencode a protein superfamily, pfam01617, which includes the predominant outer membrane proteins (OMPs) of each species, major surface protein 2 (MSP2) and MSP3 of Anaplasma marginaleand Anaplasma ovis, Anaplasma phagocytophilumMSP2 (p44), Ehrlichia chaffeensisp28-OMP, Ehrlichia canisp30, and Ehrlichia ruminantiumMAP1, and has been shown to be involved in both antigenic variation within the mammalian host and differential expression between the mammalian and arthropod hosts. Recently, complete sequencing of the A. marginalegenome has identified an expanded set of genes, designated omp1-14, encoding new members of this superfamily. Transcriptional analysis indicated that, with the exception of the three smallest open reading frames, omp2, omp3, and omp6, these superfamily genes are transcribed in A. marginale-infected erythrocytes, tick midgut and salivary glands, and the IDE8 tick cell line. OMPs 1, 4, 7 to 9, and 11 were confirmed to be expressed as proteins by A. marginalewithin infected erythrocytes, with expression being either markedly lower (OMPs 1, 4, and 7 to 9) or absent (OMP11) in infected tick cells, which reflected regulation at the transcript level. Although the pfam01617 superfamily includes the antigenically variable MSP2 and MSP3 surface proteins, analysis of the omp1-14sequences throughout a cycle of acute and persistent infection in the mammalian host and tick transmission reveals a high degree of conservation, an observation supported by sequence comparisons between the St. Maries strain and Florida strain genomes.

Numerous sheep with the prion gene (PRNP) that encodes for 171QQ develop scrapie, a neurodegenerative prion disease of sheep. Relatively few cases of scrapie-affected sheep with PRNPgenetics that encode for 171RR, however, have been found. Using flow cytometric analysis, statistically fewer PrPc-positive microglia and monocytes were observed from sheep with 171RR PRNPgenetics than from sheep with 171QQ PRNPgenetics (P<0.05). One possibility for the lack of PrPScaccumulation in brains and lymph nodes of scrapie-exposed sheep with 171RR PRNPgenetics is because of fewer PrPc-positive myeloid-derived cells available for conversion of PrPcto PrPSc.

ABSTRACTThe protozoan parasite Babesia equireplicates within erythrocytes. During the acute phase of infection, B. equican reach high levels of parasitemia, resulting in a hemolytic crisis. Horses that recover from the acute phase of the disease remain chronically infected. Subsequent transmission is dependent upon the ability of vector ticks to acquire B. equiand, following development and replication, establishment of B. equiin the salivary glands. Although restriction of the movement of chronically infected horses with B. equiis based on the presumption that ticks can acquire and transmit the parasite at low levels of long-term infection, parasitemia levels during the chronic phase of infection have never been quantified, nor has transmission been demonstrated. To address these epidemiologically significant questions, we established long-term B. equiinfections (>1 year), measured parasitemia levels over time, and tested whether nymphal Boophilus microplusticks could acquire and, after molting to the adult stage, transmit B. equito naive horses. B. equilevels during the chronic phase of infection ranged from 103.3to 106.0/ml of blood, with fluctuation over time within individual horses. B. microplusticks fed on chronically infected horses with mean parasite levels of 105.5± 100.48/ml of blood acquired B. equi, with detection of B. equiin the salivary glands of 7 to 50% of fed ticks, a range encompassing the percentage of positive ticks that had been identically fed on a horse in the acute phase of infection with high parasitemia levels. Ticks that acquired B. equifrom chronically infected horses, as well as those fed during the acute phase of infection, successfully transmitted the parasite to naive horses. The results unequivocally demonstrated that chronically infected horses with low-level parasitemia are competent mammalian reservoirs for tick transmission of B. equi.

Identifying the reservoirs of a pathogen is vital for control of sporadic disease and epidemics. Listeria monocytogenesis a zoonotic foodborne pathogen that is responsible for 28% of food-related deaths in the United States annually, as well as a major cause of massive product recalls worldwide. To examine the role of the dairy farm as a potential source or reservoir for L. monocytogenessubtypes shown to cause human listeriosis, we compared the pulsed-field gel electrophoresis (PFGE) restriction enzyme digestion profiles of L. monocytogenesdairy farm–associated strains (milk, environmental, and bovine) to human sporadic and epidemic disease strains. Twenty-three percent of human sporadic strains had PFGE patterns identical to that of farm isolate(s). Additionally, three farm environmental strains and one human sporadic strain had a PFGE pattern identical to a strain of L. monocytogenesresponsible for the 1985 California epidemic. These data indicate that this epidemic strain continues to cause sporadic human illness and has a potential dairy farm as a reservoir.

ABSTRACTEquine protozoal myeloencephalitis is a progressive neurologic disease of horses most commonly caused by infection with the apicomplexan parasite Sarcocystis neurona. Factors affecting neuroinvasion and neurovirulence have not been determined. We investigated the pathogenesis of infection with S. neuronain horses with severe combined immune deficiency (SCID). Two immunocompetent (IC) Arabian horses and two Arabian horses with SCID were infected orally with 5 × 105sporocysts of S. neurona. Four IC horses and one SCID horse were infected intravenously (i.v.) with 5 × 108merozoites of the WSU-1 isolate of S. neurona. Despite prolonged parasitemia and persistent infection of visceral tissues (skeletal muscle, cardiac muscle, lung, liver, and spleen) as demonstrated by PCR and culture, SCID horses did not develop neurologic signs after oral or i.v. infection. S. neuronawas undetectable in the neuronal tissues of SCID horses by either PCR, immunohistochemistry, or culture. In contrast, although parasitemia was undetectable in orally infected IC horses and of only short duration in i.v. infected IC horses, four of six IC horses developed neurologic signs. S. neuronawas detectable by PCR and/or culture of neural tissue but not visceral tissue of IC horses with neurologic disease. Infected SCID horses are unable to clear S. neuronafrom visceral tissues, but the infection does not result in neurologic signs; in contrast, IC horses rapidly control parasitemia and infection of visceral tissues but frequently experience neuroinvasion and exhibit clinical signs of neurologic disease.

ABSTRACTMultiple genotypically unique strains of the tick-borne pathogen Anaplasma marginaleoccur and are transmitted within regions where the organism is endemic. In this study, we tested the hypothesis that specific A. marginalestrains are preferentially transmitted. The study herd of cattle (n= 261) had an infection prevalence of 29% as determined by competitive inhibition enzyme-linked immunosorbent assay and PCR, with complete concordance between results of the two assays. Genotyping revealed the presence of 11 unique strains within the herd. Although the majority of the individuals (70 of 75) were infected with only a single A. marginalestrain, five animals each carried two strains with markedly distinct genotypes, indicating that superinfection does occur with distinct A. marginalestrains, as has been reported with A. marginaleand A. marginalesubsp. centralestrains. Identification of strains in animals born into and infected within the herd during the period from 1998 to 2003 revealed no significant difference from the overall strain prevalence in the herd, results that do not support the occurrence of preferential strain transmission within a population of persistently infected animals and are most consistent with pathogen strain transmission being stochastic.

ABSTRACTEqui merozoite antigens 1 and 2 (EMA-1 and EMA-2) are Babesia equiproteins expressed on the parasite surface during infection in horses and are orthologues of proteins in Theileriaspp., which are also tick-transmitted protozoal pathogens. We determined in this study whether EMA-1 and EMA-2 were expressed within the vector tick Boophilus microplus. B. equitransitions through multiple, morphologically distinct stages, including sexual stages, and these transitions culminate in the formation of infectious sporozoites in the tick salivary gland. EMA-2-positive B. equistages in the midgut lumen and midgut epithelial cells of Boophilus microplusnymphs were identified by reactivity with monoclonal antibody 36/253.21. This monoclonal antibody also recognized B. equiin salivary glands of adult Boophilus microplus. In addition, quantification of B. equiin the mammalian host and vector tick indicated that the duration of tick feeding and parasitemia levels affected the percentage of nymphs that contained morphologically distinct B. equiorganisms in the midgut. In contrast, there was no conclusive evidence that B. equiEMA-1 was expressed in either the Boophilus microplusmidgut or salivary gland when monoclonal antibody 36/18.57 was used. The expression of B. equiEMA-2 in Boophilus microplusprovides a marker for detecting the various development stages and facilitates the identification of novel stage-specific Babesiaproteins for testing transmission-blocking immunity.

The prevalence of Listeria monocytogenesin bulk milk from three Pacific Northwest states was assessed for 474 herds at three time points. For samples collected in November 2000 and June 2001, the L. monocytogenesprevalence levels were 4.9 and 7.0%, respectively. All isolates were subtyped by serotyping and by pulsed-field gel electrophoresis (PFGE). Forty-nine of the 55 isolates belonged to serogroup 1/2a, while 6 belonged to serogroup 4. Subtyping by PFGE revealed that isolates from 31 herds shared 10 patterns; there was a weak but significant association between PFGE subtype and geographical distance. Six herds were positive for L. monocytogenesat both time points. Of these six herds, four had indistinguishable PFGE patterns at both time points. Twenty-five of the 33 herds that were positive in June 2001 were sampled again in June 2002. L. monocytogeneswas recovered from 17 of these 25 herds (68%), with the ApaI restriction enzyme digestion profiles (REDP) for 8 herds being identical to those of isolates recovered from these herds the previous year. The ApaI REDP for the bulk milk isolates were compared with those for isolates recovered from environmental and human samples that were collected by the Washington Department of Health (n= 23). Analysis of ApaI digestion profiles revealed that only two of the Washington Department of Health isolates had digestion profiles similar to those for isolates from bulk milk; however, further analysis with the use of a second enzyme (AscI) was capable of discriminating between isolates from the two sources. Thus, we found no direct REDP matches between bulk milk and clinical isolates.

ABSTRACTWhether arthropod vectors retain competence for transmission of infectious agents in the long-term absence of vector-pathogen interaction is unknown. We addressed this question by quantifying the vector competence of two tick vectors, with mutually exclusive tropical- versus temperate-region distributions, for genetically distinct tropical- and temperate-region strains of the cattle pathogen Anaplasma marginale. The tropical cattle tick Boophilus microplus, which has been eradicated from the continental United States for over 60 years, was able to acquire and transmit the temperate St. Maries (Idaho) strain of A. marginale. Similarly, the temperate-region tick Dermacentor andersoniefficiently acquired and transmitted the Puerto Rico strain of A. marginale. There were no significant quantitative differences in infection rate or number of organisms per tick following feeding on cattle with persistent infections of either A. marginalestrain. In contrast, the significantly enhanced replication of the Puerto Rico strain in the salivary gland of B. microplusat the time of transmission feeding is consistent with adaptation of a pathogen strain to its available vector. However, the transmission of both strains by B. microplusdemonstrates that adaptation or continual interaction between the pathogen and vector is not required for retention of vector competence. Importantly, the results clearly show that reestablishment of acaricide-resistant B. microplusin the United States would be associated with A. marginaletransmission.

ABSTRACTRhodococcus equiis a gram-positive bacterium that infects alveolar macrophages and causes rhodococcal pneumonia in horses and humans. The virulence plasmid of R. equiappears to be required for both pathogenicity in the horse and the induction of protective immunity. An understanding of the mechanisms by which virulent R. equicircumvents protective host responses and by which bacteria are ultimately cleared is important for development of an effective vaccine. Six adult horses were challenged with either virulent R. equior an avirulent, plasmid-cured derivative. By using a flow cytometric method for intracytoplasmic detection of gamma interferon (IFN-?) in equine bronchoalveolar lavage fluid (BALF) cells, clearance of the virulent strain was shown to be associated with increased numbers of pulmonary CD4+and CD8+T lymphocytes producing IFN-?. There was no change in IFN-?-positive cells in peripheral blood, suggesting that a type 1 recall response at the site of challenge was protective. The plasmid-cured strain of R. equiwas cleared in horses without a significant increase in IFN-?-producing T lymphocytes in BALF. In contrast to these data, a previous report in foals suggested an immunomodulating role for R. equivirulence plasmid-encoded products in downregulating IFN-? expression by equine CD4+T lymphocytes. Intracytoplasmic detection of IFN-? provides a method to better determine whether modulation of macrophage-activating cytokines by virulent strains occurs uniquely in neonates and contributes to their susceptibility to rhodococcal pneumonia.

ABSTRACTEqui merozoite antigen 1 (EMA-1) is an immunodominant Babesia equierythrocyte-stage surface protein. A competitive enzyme-linked immunosorbent assay (ELISA), based on inhibition of monoclonal antibody (MAb) 36/133.97 binding to recombinant EMA-1 by equine anti-B. equiantibodies, detects horses infected with strains present throughout the world. The objectives of this study were to define the epitope bound by MAb 36/133.97 and quantify the amino acid conservation of EMA-1, including the region containing the epitope bound by MAb 36/133.97. The alignment of the deduced amino acid sequence of full-length EMA-1 (Florida isolate) with 15 EMA-1 sequences from geographically distinct isolates showed 82.8 to 99.6% identities (median, 98.5%) and 90.5 to 99.6% similarities (median, 98.9%) between sequences. Full-length and truncated recombinant EMA-1 proteins were expressed and tested for their reactivities with MAb 36/133.97. Binding required the presence of amino acids on both N- and C-terminal regions of a truncated peptide (EMA-1.2) containing amino acids 1 to 98 of EMA-1. This result indicated that the epitope defined by MAb 36/133.97 is dependent on conformation. Sera from persistently infected horses inhibited the binding of MAb 36/133.97 to EMA-1.2 in a competitive ELISA, indicating that equine antibodies which inhibit binding of MAb 36/133.97 also recognize epitopes in the same region (the first 98 residues). Within this region, the deduced amino acid sequences had 85.7 to 100% identities (median, 99.0%), with similarities of 94.9 to 100% (median, 100%). Therefore, the region which binds to both MAb 36/133.97 and inhibiting equine antibodies has a median amino acid identity of 99.0% and a similarity of 100%. These data provide a molecular basis for the use of both EMA-1 and MAb 36/133.97 for the detection of antibodies against B. equi.

ABSTRACTPeripheral blood leukocytes (PBLs) from scrapie-infected sheep were evaluated for the presence of PrPScby using dissociated retropharyngeal lymph node (DRLN) cells and immunohistochemistry (IHC). PrPSc-positive cells were detected in 2.05% ± 0.28% of 3 × 106DRLN cells, but were not detected in 3 × 106PBLs from scrapie-infected sheep. Titration of DRLN cells mixed with PBLs showed that IHC detects a minimum of 0.00205% or 60 PrPSc-positive cells in 3 × 106PBLs.

ABSTRACTTo investigate the in vivo role of CD4+T lymphocytes during acute anaplasmosis, thymectomized calves were selectively depleted of CD4+T lymphocytes by treatment with anti-CD4 monoclonal antibody (MAb) and were then infected with the Florida strain of Anaplasma marginalein two sequential experiments (experiments 1 and 2). Treatment of thymectomized calves with a total of 5.0 mg of anti-CD4 MAb/kg of body weight during the 1st week followed by 0.3-mg/kg doses administered twice weekly for 7 weeks resulted in significant depletion of CD3+CD4+and CD4+CD45R+(naive) T lymphocytes from blood, spleen, and peripheral lymph nodes for the duration of the 8-week study, compared to the results for thymectomized control calves treated with a subclass-matched MAb. All calves became parasitemic and pyretic following experimental infection with A. marginale, and decreases in packed cell volume (PCV) coincided with peak parasitemia. No significant differences in PCV or parasitemia were observed between treatment groups. Thymectomized calves treated with anti-CD4 MAb were able to mount an anti-A. marginaleantibody response, although in experiment 2, anti-CD4 MAb-treated calves had four- to sixfold lower immunoglobulin G1 (IgG1) and no detectable IgG2 anti-A. marginalemajor surface protein 2-specific antibody titers compared to thymectomized control calves treated with a subclass-matched MAb. At the level of CD4+-T-lymphocyte depletion achieved and experimental anaplasmosis induced, thymectomized anti-CD4 MAb-treated calves were able to control acute anaplasmosis. This was in contrast to the prediction that significant depletion of CD4+T lymphocytes would abrogate resistance to acute infection.

Peripheral blood leukocytes (PBLs) from scrapie-infected sheep were evaluated for the presence of PrP(Sc) by using dissociated retropharyngeal lymph node (DRLN) cells and immunohistochemistry (IHC). PrP(Sc)-positive cells were detected in 2.05% +/- 0.28% of 3 x 10(6) DRLN cells, but were not detected in 3 x 10(6) PBLs from scrapie-infected sheep. Titration of DRLN cells mixed with PBLs showed that IHC detects a minimum of 0.00205% or 60 PrP(Sc)-positive cells in 3 x 10(6) PBLs.

ABSTRACTA competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific forBabesia caballi. The assay used recombinant B. caballirhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballiantigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballiby a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive with B. caballiRAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive withB. caballiwhen they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballiantibodies.

ABSTRACTImmunization with the native major surface protein 1 (MSP1) (a heterodimer containing disulfide and noncovalently bonded polypeptides designated MSP1a and MSP1b) of the erythrocytic stage ofAnaplasma marginaleconferred protection against homologous challenge (G. H. Palmer, A. F. Barbet, W. C. Davis, and T. C. McGuire, Science 231:1299–1302, 1986). The MSP1a polypeptide possesses a conserved neutralization-sensitive epitope. In the present study, the immune response to DNA-mediated immunization using msp1a was studied. The plasmid pVCL/MSP1a, which encodes the complete msp1a gene of A. marginaleunder the control of human cytomegalovirus immediate-early enhancer/promoter and intron A, was constructed. The immune responses elicited by immunization with pVCL/MSP1a into cardiotoxin-induced regenerating muscle were evaluated in mice and cattle. Antibody reactive with native MSP1a was detected in pooled sera of immunized BALB/c mice 3 weeks following primary immunization. Two calves seronegative for A. marginalewere immunized four times, at weeks 0, 3, 7, and 13, with pVCL/MSP1a. By 8 weeks, both calves responded to MSP1a with an antibody titer of 1:100, which peaked at 1:1,600 and 1:800 by 16 weeks after the initial immunization. Interestingly, immunoblotting with anti-immunoglobulin G1 (anti-IgG1) and anti-IgG2 specific monoclonal antibodies revealed a restricted IgG1 anti-MSP1a response in both animals. T-lymphocyte lines, established after the fourth immunization, proliferated specifically againstA. marginalehomogenate and purified MSP1 in a dose-dependent manner. These data provide a basis for an immunization strategy to direct bovine immune responses by using DNA vaccine vectors containing single or multiple genes encoding major surface proteins ofA. marginale.

ABSTRACTA competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginalewas validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginaleis endemic. The true positive and negative A. marginaleinfection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale. Third, msp5sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale. The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginaleinfections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement.

ABSTRACTThe Anaplasma marginaleouter membrane is composed of immunogenic major surface proteins (MSPs) linked both covalently and noncovalently in multimeric complexes (M. C. Vidotto, T. C. McGuire, T. F. McElwain, G. H. Palmer, and D. P. Knowles, Infect. Immun. 62:2940–2946). Consequently, effective induction of antibody against surface-exposed MSP epitopes has been postulated to require maintenance of MSP secondary through quatenary structures. Using MSP5 as a model and the approach of epitope mapping with recombinant expressed full-length and truncated proteins, we demonstrated that the immunodominant surface epitope bound by monoclonal antibody (MAb) ANAF16C1 required disparate amino- and carboxy-terminal regions of MSP5, indicating the conformational dependence of this epitope. The required amino-terminal MSP5 region included the cysteines involved in intramolecular disulfide bonding. The dependence of the immunodominant epitope on disulfide bonding was confirmed by loss of MAb ANAF16C1 binding to MSP5 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. The recognition of the MSP5 immunodominant epitope by antibody induced by protective immunization with A. marginaleouter membranes was also conformationally dependent, as shown by the loss of epitope binding following serum adsorption with native but not reduced and denaturedA. marginale. Importantly, the antibody response to all immunodominant MSP5 surface epitopes was restricted to conformationally dependent epitopes, since the binding of polyclonal anti-MSP5 antibody to the A. marginalesurface could be blocked by adsorption with native but not denatured and reduced MSP5. These results confirm the importance of the secondary and tertiary structures of MSP epitopes as immune system targets and support the testing of immunogens which maintain the required conformation.

A highly specific and sensitive competitive enzyme-linked immunosorbent assay for detection of specific antibody to Babesia equiin serum from equids was validated for use in Morocco. The assay is based on the specific inhibition of binding of a monoclonal antibody to a conserved epitope within a recombinant parasite peptide by serum from infected animals. The assay was compared to an established indirect immunofluorescence assay, with a concordance of 91%. The assay was used to determine seroprevalence for B. equiinfections in donkeys and horses throughout Morocco. A total of 578 sera (163 horses and 415 donkeys) from 6 locations representing different bioclimatic regions were assayed. An analysis of variance, indicated no significant effect of location; however, donkeys were significantly more likely than horses to be seropositive. Management conditions contribute to greater tick infestations and thus Babesiaexposure in donkeys than in horses.

Theileria parvais the causative agent of East Coast fever (ECF), a tick-borne disease that kills over a million cattle each year in sub-Saharan Africa. Immune protection against T. parvainvolves a CD8+cytotoxic T cell response to parasite-infected cells. However, there is currently a paucity of knowledge regarding the role played by innate immune cells in ECF pathogenesis and T. parvacontrol.

Fifteen newborn Saanen goats were orally infected with a biologically cloned isolate of caprine arthritis-encephalitis virus (CAEV-63). At 33.5 months after infection, 8 of 12 seropositive goats had CAEV-63-specific neutralizing antibody. Five neutralization-positive goats developed clinically apparent arthritis of carpal joints, three of which had periarticular swelling indicating severe synovitis. Arthritis was not evident in age-matched controls or infected goats without neutralizing antibody. Multiple viral isolates were obtained from synovial fluid or synovial fluid cells ofarthritic joints between 36.4 and 44.9 months after infection, and successive isolates from individual joints were defined as antigenic variants expressing type-specific neutralization epi-topes. Thus, the evolution of neutralizing antibody does not arrest viral replication or development ofprogressive inflammatory lesions. Rather, some antigenic variants produced in the presence of neutralizing antibody are clonally expanded within carpal joints, and severe joint inflammation is specifically associated with these variants.