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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.

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.

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.

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.

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.

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.

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.