Evaluating Two Sampling Methods for Mycoplasma Bovis Diagnosis in American Bison (Bison Bison).

Mycoplasma bovis is a bacterial pathogen endemic to cattle. In the early 2000s, M. bovis emerged as a cause of respiratory disease in American bison (Bison bison), causing significant morbidity and mortality. Bison herds that experience an outbreak of M. bovis are at higher risk for subsequent outbreaks, suggesting that chronic, subclinical infections can be established. Antemortem testing is therefore crucial to disease management; however, the precise sampling method to maximize detection of M. bovis in bison is unknown. We evaluated two sample types-superficial nasal swabs and deep nasopharyngeal swabs-collected from apparently healthy or symptomatic bison from January 2021 through December 2022. We used real-time PCR to detect M. bovis in 76/938 bison (8.1%) from 11 herds. For bison testing positive on at least one swab type, M. bovis was detected in 63/76 (82.8%) deep nasopharyngeal swabs and 29/73 (38.1%) superficial nasal swabs. Agreement between swabs for positive bison was 21% (n=16, kappa coefficient 0.319). We conclude that deep nasopharyngeal swabbing is more sensitive than superficial nasal swabbing for detection of M. bovis in bison and that low agreement between methods may be related to stage of infection. We further tested pooled samples by PCR and found that pooling of up to five samples can be effective to increase throughput and minimize costs. Management of wild bison relies on the ability to relocate animals to maintain gene flow and healthy populations. Sensitive and specific diagnostic tests are needed to inform decisions and minimize risk of transmission, especially from subclinical carriers. This study provides valuable insight that will inform best practices for M. bovis testing, thereby supporting the conservation of bison as healthy wildlife, which in turn promotes ecological restoration, safeguards cultural practices of Tribal Nations, and upholds the bison as a unique American icon.

Serum IgG Immunoglobulin Levels are Associated with Reduced PCR Detection of Mycoplasma bovis in Naturally Infected American Bison (Bison bison).

Mycoplasma bovis is an important pathogen of North American bison (Bison bison), associated with high morbidity and mortality epizootics of respiratory and reproductive disease. Despite the significant negative impact on bison health, little is known about the kinetics of disease and the host immune response to infection. To address these questions, a cohort of bison calves was created and serially sampled 5 times, once every 2-3 mo, over a 12-mo period. At each sampling period nasal swab samples were collected and tested by PCR for the presence of M. bovis. Serum samples were also collected and assessed for M. bovis-specific antibodies using both a commercial and an in-house ELISA. Overall, 19/41 bison (46.3%) had positive PCR tests, and 31/41 (75.6%) were seropositive. Over the course of the study, the frequency of PCR-positive nasal swabs and the ELISA scores decreased, although serum samples remained positive for at least 6 mo following the final positive PCR test. Bison were grouped according to results from the in-house ELISA into high-responder (n=7), low-responder (n=5), and seronegative (n=7) groups. Mycoplasma bovis-specific IgG antibody levels were significantly elevated in the high-responder group compared to the low-responder and seronegative groups. The differences were statistically significant for 3/5 sampling periods. A trend toward increased IgG2 levels was observed in the high-responder group. High total IgG responses correlated with a decline in positive PCR tests from nasal swabs. These data provide evidence that a strong humoral response is beneficial and is probably involved in the clearance of M. bovis from bison.

NOVEL AVIBACTERIUM SPECIES ASSOCIATED WITH SINUSITIS AND CONJUNCTIVITIS IN A MERRIAM'S WILD TURKEY (MELEAGRIS GALLOPAVO MERRIAMI) FLOCK IN COLORADO, USA.

A Merriam's Wild Turkey (Meleagris gallopavo merriami) with periocular swelling and periocular skin crusting in Pueblo County, Colorado, USA, was diagnosed with severe catarrhal and fibrinous sinusitis and conjunctivitis. A novel clade of Avibacterium was detected in the exudate from this bird. Although eight additional turkeys culled from the affected flock did not have clinical signs or gross lesions, histologically all had mild-to-moderate chronic sinusitis, and infraorbital cultures yielded the same novel clade of Avibacterium that was found in the symptomatic turkey. The presence of this Avibacterium species in the absence of significant disease in some birds suggested that other factors might have been involved in the development of severe sinusitis and conjunctivitis in the symptomatic Wild Turkey. Negative culture results from a distant flock of Wild Turkeys, acquired with similar methods to the affected flock, suggested that this novel species of Avibacterium was not widespread throughout Wild Turkeys in Colorado.

SOURCE AND SEASONALITY OF EPIZOOTIC MYCOPLASMOSIS IN FREE-RANGING PRONGHORN (ANTILOCAPRA AMERICANA).

Mycoplasma bovis is an economically important bacterial pathogen of cattle (Bos taurus) and bison (Bison bison) that most commonly causes pneumonia, polyarthritis, and mastitis. It is prevalent in cattle and ranched bison; however, infections in other species are rare. In early 2019, we identified M. bovis in free-ranging pronghorn (Antilocapra americana) in northeastern Wyoming. Here, we report on additional pronghorn mortalities caused by M. bovis, in the same approximately 120-km2 geographic region 1 yr later. Genetic analysis by multilocus sequence typing revealed that the mortalities were caused by the same M. bovis sequence type, which is unique among all sequence types documented thus far in North America. To explore whether pronghorn maintain chronic infections and begin assessing M. bovis status in other sympatric species, we used PCR testing of nasal swabs to opportunistically survey select free-ranging ungulates. We found no evidence of subclinical infections in 13 pronghorn sampled from the outbreak area (upper 95% binomial confidence limit [bCL], ∼24.7%) or among 217 additional pronghorn (upper 95% bCL, ∼1.7%) sampled from eight additional counties in Wyoming and 10 in Montana. All mule deer (Odocoileus hemionus; n=231; upper 95% bCL, ∼1.6%) sampled from 11 counties in Wyoming also were PCR negative. To assess the potential for environmental transmission, we examined persistence of M. bovis in various substrates and conditions. Controlled experiments revealed that M. bovis can remain viable for 6 h in shaded water and 2 h in direct sunlight. Our results indicate that environmental transmission of M. bovis from livestock to pronghorn is possible and that seasonality of infection could be due to shared resources during late winter. Further investigations to better understand transmission dynamics, to assess population level impacts to pronghorn, and to determine disease risks among pronghorn and other ungulate taxa appear warranted.

Fatal Chlamydia psittaci infection in a domestic kitten.

Chlamydia psittaci has not been reported to cause disease in domestic cats, to our knowledge. In contrast, C. felis infection is common in domestic cats and typically results in conjunctivitis, upper respiratory tract infection, and less frequently pneumonia. Herein, we report the pathologic findings and diagnostic features of a fatal case of psittacosis in a 7-wk-old domestic kitten. The animal was 1 of a litter of 5 that, together with the queen, were yielded to a pet rescue center in Wyoming. Over a period of ~3 wk, the kittens and queen became sick, thin, and icteric prior to death, despite antimicrobial treatments. Postmortem evaluation of a kitten revealed necrosuppurative hepatitis with Gimenez stain-positive intracellular bacteria, nonsuppurative pneumonia, and mild leptomeningitis. The diagnosis of psittacosis was made by 16S rRNA PCR using multiple primer sets and sequencing from liver. Psittacosis should be considered a differential diagnosis in domestic cats with intracellular bacterial hepatitis and interstitial pneumonia.

Mycoplasma bovis Infections in Free-Ranging Pronghorn, Wyoming, USA.

Mycoplasma bovis is 1 of several bacterial pathogens associated with pneumonia in cattle. Its role in pneumonia of free-ranging ungulates has not been established. Over a 3-month period in early 2019, ¼60 free-ranging pronghorn with signs of respiratory disease died in northeast Wyoming, USA. A consistent finding in submitted carcasses was severe fibrinosuppurative pleuropneumonia and detection of M. bovis by PCR and immunohistochemical analysis. Multilocus sequence typing of isolates from 4 animals revealed that all have a deletion in 1 of the target genes, adh-1. A retrospective survey by PCR and immunohistochemical analysis of paraffin-embedded lung from 20 pronghorn that died with and without pneumonia during 2007-2018 yielded negative results. These findings indicate that a distinct strain of M. bovis was associated with fatal pneumonia in this group of pronghorn.

Comparison of 2 ELISAs for detecting exposure to Brucella ovis.

Control of Brucella ovis infection in sheep flocks in the United States depends on early detection of B. ovis antibodies via serologic testing. We used 2,276 sheep sera and various cutoff values to compare seroprevalence and agreement between 2 ELISAs: the National Veterinary Services Laboratories (NVSL) B. ovis indirect ELISA and the IDEXX B. ovis ELISA kit. A subset of 295 sera was used to compare agreement and evaluate relative sensitivity and specificity of the 2 ELISAs with an agar gel immunodiffusion (AGID) test kit. There was no significant difference in B. ovis seroprevalence between the ELISAs; however, there was poor agreement between them. When the AGID test was used as the reference test, the IDEXX ELISA with a moderate cutoff value (S/P ratio = 45%) had the highest relative sensitivity of 38.1% and specificity of 92.0%. The NVSL ELISA with a lax cutoff value (S/P ratio = 0.75) had relative sensitivity of 19.1% and specificity of 94.6%. Receiver operating characteristic analysis revealed that optimal cutoff values for the NVSL and IDEXX ELISAs were 0.091 and 16.5%, respectively. This results in sensitivity and specificity of 85.7% and 31.8% for the NVSL ELISA, and sensitivity and specificity of 81.0% and 53.6% for the IDEXX ELISA, respectively.

City sanitation and socioeconomics predict rat zoonotic infection across diverse neighbourhoods.

Rat-associated zoonoses transmitted through faeces or urine are of particular concern for public health because environmental exposure in homes and businesses may be frequent and undetected. To identify times and locations with greater public health risks from rats, we investigated whether rat characteristics, environmental features, socioeconomic factors, or season could predict rat infection risk across diverse urban neighbourhoods. In partnership with a pest management company, we sampled rats in 13 community areas along an income gradient in Chicago, a large city where concern about rats has increased in recent years. We collected kidneys for Leptospira spp. testing and colon contents for aerobic bacteria such as Salmonella spp. and Escherichia coli. Of 202 sampled rats, 5% carried Leptospira spp. and 22% carried E. coli. Rats were significantly more likely to carry Leptospira spp. on blocks with more standing water complaints in higher-income neighbourhoods (OR = 6.74, 95% CI: 1.54-29.39). Rats were significantly more likely to carry E. coli on blocks with more food vendors (OR = 9.94, 2.27-43.50) particularly in low-income neighbourhoods (OR = 0.26, 0.09-0.82) and in the spring (OR = 15.96, 2.90-88.62). We detected a high diversity of E. coli serovars but none contained major virulence factors. These associations between environmental features related to sanitation and infection risk in rats support transmission through water for Leptospira spp. and faecal-oral transmission for E. coli. We also found opposing relationships between zoonotic infection risk and income for these two pathogens. Thus, our results highlight the importance of sanitation for predicting zoonotic disease risks and including diverse urban areas in pathogen surveillance to mitigate public health risks from rats.

Seroprevalence and risk factors of Brucella ovis in domestic sheep in Wyoming, USA.

BACKGROUND: Brucella ovis causes a sexually transmitted, infectious disease of domestic sheep characterized by genital lesions and epididymitis in rams, placentitis and rare abortions in ewes, and neonatal mortality in lambs. This study was designed to 1) estimate animal and flock seroprevalence of B. ovis in sheep across Wyoming, USA, and 2) describe epidemiologic risk factors associated with seropositive sheep and flocks. For the animal seroprevalence estimate, 2423 blood samples were collected from sheep on 18 producer-selected operations and a questionnaire about possible risk factors was distributed. For the flock seroprevalence estimate, blood samples from 82 operations were obtained, including samples from the previous 18 operations and 64 additional operations that sent samples to the Wyoming State Veterinary Laboratory for diagnostic testing. Categorical risk factors were created based on questionnaires and submission forms. Sera was analyzed using the B. ovis enzyme-linked immunosorbent assay. RESULTS: Estimated true animal and flock seroprevalence were 0.53% (95% CI: 0.21-1.01%; 22/2,423) and 22.5% (95% CI: 14-32%; 18/82), respectively. Using Fisher's exact and Mid-p exact tests to compare apparent seroprevalence with respect to possible risk factors, increased age and breed type were risk factors associated with seropositive sheep, while region and large flock size were risk factors associated with seropositive flocks. CONCLUSIONS: Results from this study suggest few sheep have been exposed to B. ovis, but many flocks contain at least one seropositive animal. Each region in Wyoming contained at least one seropositive animal and flock, emphasizing the importance of disease-free documentation before purchasing new sheep. Aged sheep (≥ 6 years of age) had the highest seroprevalence among age groups; hence, we propose the separation of young rams from older rams to help reduce disease spread outside the breeding season. Wool breeds (Rambouillet and Merino) may be less susceptible to B. ovis infection given they had the lowest animal seroprevalence of the breed types, and large flocks (> 100 breeding rams) had the highest seroprevalence of the flock size categories, likely due to more intensive management strategies that can contribute to the introduction and persistence of B. ovis infection in sheep and flocks.

Bovine abortion caused by Coxiella burnetii: report of a cluster of cases in Uruguay and review of the literature.

A cluster of 4 bovine abortions caused by Coxiella burnetii occurred in a dairy herd in Uruguay during a 2-mo period. Case 1 consisted of a placenta from an aborted cow; cases 2-4 were fetuses and their placentas. Grossly, the placenta from one aborted cow had moderate, diffuse reddening of the cotyledons and loss of translucency of the intercotyledonary areas. No gross lesions were observed in the other 3 placentas. Microscopically, 2 of 4 placentas had fibrinonecrotizing placentitis with abundant intratrophoblastic gram-negative coccobacilli. C. burnetii was identified intralesionally by immunohistochemistry (IHC) in all 4 placentas, and by PCR and DNA sequencing in 3 placentas analyzed by these techniques. One fetus had mild neutrophilic alveolitis with multinucleate syncytial cells; no gross or microscopic lesions were observed in the other 2 fetuses examined. The lungs of the 3 fetuses were negative for C. burnetii by IHC. Tests performed to investigate other possible causes of abortions in the 4 cases were negative. C. burnetii causes Q fever in humans and coxiellosis in animals. Clusters of abortions in cattle by C. burnetii have not been reported previously, to our knowledge; this bacterium has been considered an opportunistic pathogen associated only with sporadic abortion in cattle. We present herein a cluster of 4 bovine abortions caused by C. burnetii in a dairy farm during a period of 2 mo and a review of the literature on C. burnetii infection in cattle.

Innate Lymphoid Cells in Protection, Pathology, and Adaptive Immunity During Apicomplexan Infection.

Apicomplexans are a diverse and complex group of protozoan pathogens including Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., Eimeria spp., and Babesia spp. They infect a wide variety of hosts and are a major health threat to humans and other animals. Innate immunity provides early control and also regulates the development of adaptive immune responses important for controlling these pathogens. Innate immune responses also contribute to immunopathology associated with these infections. Natural killer (NK) cells have been for a long time known to be potent first line effector cells in helping control protozoan infection. They provide control by producing IL-12 dependent IFNγ and killing infected cells and parasites via their cytotoxic response. Results from more recent studies indicate that NK cells could provide additional effector functions such as IL-10 and IL-17 and might have diverse roles in immunity to these pathogens. These early studies based their conclusions on the identification of NK cells to be CD3-, CD49b+, NK1.1+, and/or NKp46+ and the common accepted paradigm at that time that NK cells were one of the only lymphoid derived innate immune cells present. New discoveries have lead to major advances in understanding that NK cells are only one of several populations of innate immune cells of lymphoid origin. Common lymphoid progenitor derived innate immune cells are now known as innate lymphoid cells (ILC) and comprise three different groups, group 1, group 2, and group 3 ILC. They are a functionally heterogeneous and plastic cell population and are important effector cells in disease and tissue homeostasis. Very little is known about each of these different types of ILCs in parasitic infection. Therefore, we will review what is known about NK cells in innate immune responses during different protozoan infections. We will discuss what immune responses attributed to NK cells might be reconsidered as ILC1, 2, or 3 population responses. We will then discuss how different ILCs may impact immunopathology and adaptive immune responses to these parasites.

Evaluation of serologic testing of rams in the management of Brucella ovis in a domestic sheep flock.

Brucella ovis is a bacterial pathogen present in most major sheep-producing regions of the world. The pathogen is associated with ram infertility, decreased ewe conception rates, and premature lambs. Twenty ELISA seropositive or indeterminate rams were culled from a B. ovis-positive flock, and donated to the Wyoming State Veterinary Laboratory for evaluation of infection. Tissues from each ram were collected at autopsy for additional testing, including bacterial culture, PCR, and histopathology. Of 17 seropositive rams, 11 rams were also positive by culture and PCR, and had evidence of mild histologic lesions; 1 seropositive ram was positive by culture with mild histologic lesions, but negative by PCR. Five seropositive rams were negative by culture and PCR and had no histologic lesions. Three indeterminate rams were negative by culture and by PCR and had no histologic lesions. The tissues in which B. ovis was most often detected included the epididymis, vesicular gland, and ampulla. Although this was a small study, the observation that 5 of 17 (29%) rams that were initially seropositive had no evidence of infection is interesting. A convalescent test for valuable seropositive animals prior to culling may be useful, and reproductive tissues may be evaluated postmortem if confirmatory testing is desired.

Infectious keratoconjunctivitis in free-ranging mule deer in Wyoming: a retrospective study and identification of a novel alphaherpesvirus.

We describe the clinicopathologic findings, relative prevalence, and pathogens associated with infectious keratoconjunctivitis in mule deer ( Odocoileus hemionus) in Wyoming. Seventeen cases with ocular lesions were identified among 1,036 mule deer postmortem submissions (1.6%) in an ~16 y period. Sixteen cases were observed in winter and most were in male (15 cases) and juvenile (13 cases) deer. Blindness was the most commonly reported clinical sign (10 cases). A herpesvirus was detected only in the 4 cases of bilateral necrotizing bulbar conjunctivitis. Phylogenetic analysis of glycoprotein amino acid sequences consistently identified this virus as a novel alphaherpesvirus. In 2 of these herpesvirus-positive cases, Actinomyces sp. and Moraxella ovis were also identified. Trueperella pyogenes was identified in 4 cases of unilateral ulcerative keratitis, keratoconjunctivitis, and panophthalmitis. M. ovis was cultured from 3 cases of bilateral conjunctivitis and keratoconjunctivitis. In the remaining cases, isolates included Moraxella bovis (1 case), Staphylococcus sp. and Streptococcus sp. (2), Flavobacterium sp. and Pseudomonas sp. (2), Escherichia coli and Enterobacter sp. (1), and bovine viral diarrhea virus 1 (1). No pathogens were identified in 2 cases. The relative prevalence of keratoconjunctivitis in mule deer in Wyoming appears to be low, and this disease is most commonly associated with infection by a novel alphaherpesvirus, T. pyogenes, and M. ovis.

Development and validation of a real-time PCR specific for the leukotoxin gene of Bibersteinia trehalosi.

A real-time PCR assay for the leukotoxin gene of Bibersteinia trehalosi was developed and validated to better identify this pathogen, which is a cause of respiratory disease in bighorn sheep. The specificity of the PCR primers was evaluated with DNA from 59 known isolates of the Pasteurellaceae family. For validation, 162 field samples were compared using both the new assay and an indirect method using 2 sets of published protocols. The real-time PCR assay was found to be specific for the leukotoxin gene of B. trehalosi and provides a rapid and direct approach for detecting leukotoxin-producing forms of this organism from samples containing mixed species of leukotoxin-positive Pasteurellaceae.

Tick passage results in enhanced attenuation of Babesia bovis.

Serial blood passage of virulent Babesia bovis in splenectomized cattle results in attenuated derivatives that do not cause neurologic disease. Tick transmissibility can be lost with attenuation, but when retained, attenuated B. bovis can revert to virulence following tick passage. This study provides data showing that tick passage of the partially attenuated B. bovis T2Bo derivative strain further decreased virulence compared with intravenous inoculation of the same strain in infected animals. Ticks that acquired virulent or attenuated parasites by feeding on infected cattle were transmission fed on naive, splenectomized animals. While there was no significant difference between groups in the number of parasites in the midgut, hemolymph, or eggs of replete female ticks after acquisition feeding, animals infected with the attenuated parasites after tick transmission showed no clinical signs of babesiosis, unlike those receiving intravenous challenge with the same attenuated strain prior to tick passage. Additionally, there were significantly fewer parasites in blood and tissues of animals infected with tick-passaged attenuated parasites. Sequencing analysis of select B. bovis genes before and after tick passage showed significant differences in parasite genotypes in both peripheral blood and cerebral samples. These results provide evidence that not only is tick transmissibility retained by the attenuated T2Bo strain, but also it results in enhanced attenuation and is accompanied by expansion of parasite subpopulations during tick passage that may be associated with the change in disease phenotype.

Comparative transcriptome analysis of geographically distinct virulent and attenuated Babesia bovis strains reveals similar gene expression changes through attenuation.

BACKGROUND: Loss of virulence is a phenotypic adaptation commonly seen in prokaryotic and eukaryotic pathogens. This mechanism is not well studied, especially in organisms with multiple host and life cycle stages such as Babesia, a tick-transmitted hemoparasite of humans and animals. B. bovis, which infects cattle, has naturally occurring virulent strains that can be reliably attenuated in vivo. Previous studies suggest the virulence loss mechanism may involve post-genomic modification. We investigated the transcriptome profiles of two geographically distinct B. bovis virulent and attenuated strain pairs to better understand virulence loss and to gain insight into pathogen adaptation strategies. RESULTS: Expression microarray and RNA-sequencing approaches were employed to compare transcriptome profiles of two B. bovis strain pairs, with each pair consisting of a virulent parental and its attenuated derivative strain. Differentially regulated transcripts were identified within each strain pair. These included genes encoding for VESA1, SmORFs, undefined membrane and hypothetical proteins. The majority of individual specific gene transcripts differentially regulated within a strain were not shared between the two strains. There was a disproportionately greater number of ves genes upregulated in the virulent parental strains. When compared with their attenuated derivatives, divergently oriented ves genes were included among the upregulated ves genes in the virulent strains, while none of the upregulated ves genes in the attenuated derivatives were oriented head to head. One gene family whose specific members were consistently and significantly upregulated in expression in both attenuated strains was spherical body protein (SBP) 2 encoding gene where SBP2 truncated copies 7, 9 and 11 transcripts were all upregulated. CONCLUSIONS: We conclude that ves heterodimer pair upregulation and overall higher frequency of ves gene expressions in the virulent strains is consistent with the involvement of this gene family in virulence. This is logical given the role of VESA1 proteins in cytoadherence of infected cells to endothelial cells. However, upregulation of some ves genes in the attenuated derivatives suggests that the consequence of upregulation is gene-specific. Furthermore, upregulation of the spherical body protein 2 gene family may play a role in the attenuated phenotype. Exactly how these two gene families may contribute to the loss or gain of virulence is discussed.

Seroprevalence of Coxiella burnetii in Washington State domestic goat herds.

A caprine herd seroprevalence of Coxiella burnetii infection was determined by passive surveillance of domestic goat herds in Washington State. Serum samples (n=1794) from 105 herds in 31 counties were analyzed for C. burnetii antibodies using a commercially available Q fever antibody enzyme-linked immunosorbent assay (ELISA) test kit. The sera were submitted to the Washington Animal Disease Diagnostic Laboratory for routine serologic screening over an approximate 1-year period from November, 2010, through November, 2011. To avoid bias introduced by testing samples from ill animals, only accessions for routine screening of nonclinical animals were included in the study. A standard cluster sampling approach to investigate seroprevalence at the herd level was used to determine optimal study sample size. The results identified C. burnetii antibodies in 8.0% of samples tested (144/1794), 8.6% of goat herds tested (9/105), and 25.8% of counties tested (8/31). Within-herd seroprevalence in positive counties ranged from 2.9% to 75.8%. Counties with seropositive goats were represented in the western, eastern, southeastern, and Columbia basin agricultural districts of the state. To our knowledge this is the first county-specific, statewide study of C. burnetii seroprevalence in Washington State goat herds. The findings provide baseline information for future epidemiologic, herd management and public health investigations of Q fever.

Loss of neurovirulence is associated with reduction of cerebral capillary sequestration during acute Babesia bovis infection.

BACKGROUND: Severe neurological signs that develop during acute infection by virulent strains of Babesia bovis are associated with sequestration of infected erythrocytes in cerebral capillaries. Serial passage of virulent strains in cattle results in attenuated derivatives that do not cause neurologic disease. We evaluated whether serial passage also results in a loss of cerebral capillary sequestration by examining brain biopsies during acute disease and at necropsy. FINDINGS: Cerebral biopsies of spleen intact calves inoculated intravenously with a virulent or attenuated strain pair of B. bovis were evaluated for capillary sequestration at the onset of babesiosis and during severe disease. In calves infected with the virulent strain, there was a significant increase in sequestration between the first and second biopsy timepoint. The attenuated strain was still capable of sequestration, but at a reduced level, and did not change significantly between the first and second biopsy. Necropsy examination confirmed the second biopsy results and demonstrated that sequestration identified at necropsy reflects pathologic changes occurring in live animals. CONCLUSIONS: Loss of neurovirulence after serial in vivo passage of the highly virulent T2Bo strain of B. bovis in splenectomized animals is associated with a significant reduction of cerebral capillary sequestration. Previous genomic analysis of this and two other strain pairs suggests that this observation could be related to genomic complexity, particularly of the ves gene family, rather than consistent gene specific differences. Additional experiments will examine whether differential gene expression of ves genes is also associated with reduced cerebral sequestration and neurovirulence in attenuated strains.

Acute and persistent infection by a transfected Mo7 strain of Babesia bovis.

A Mo7-derived Babesia bovis line stably transfected with the gfp-bsd gene was inoculated into two four to five months old calves, while two additional calves were inoculated with Mo7 parasites. Similar mild clinical signs were detected in all calves. B. bovis rap-1 was identified in the bloodstream by PCR four days post inoculation (dpi), and consistently over ten months thereafter. Transfusion of blood from experimentally infected calves into four naïve splenectomized calves at 212 dpi resulted in acute disease in recipients, confirming persistent infection in the four donor animals. The proportion of GFP expressing parasites recovered from a splenectomized recipient calf is undistinguishable from transfected parasites that were maintained in long term culture under blasticidin selection. Furthermore, the sequences of transfected genes in recovered parasites remained unaltered. Together, the data demonstrates that exogenous B. bovis transgenes can be expressed and remain stable throughout acute and persistent infection in calves.

Expression and strain variation of the novel "small open reading frame" (smorf) multigene family in Babesia bovis.

Small open reading frame (smorf) genes comprise the second largest Babesia bovis multigene family. All known 44 variant smorf genes are located in close chromosomal proximity to ves1 genes, which encode proteins that mediate cytoadhesion and contribute to immune evasion. In this study, we characterised the general topology of smorf genes and investigated the gene repertoire, transcriptional profile and SMORF expression in two distinct strains, T2Bo and Mo7. Sequence analysis using degenerate primers identified additional smorf genes in each strain and demonstrated that the smorf gene repertoire varies between strains, with conserved and unique genes in both. Smorf genes have multiple semi-conserved and variable blocks, and a large hypervariable insertion in 20 of the 44 genes defines two major branches of the family, termed smorf A and smorf B. A total of 32 smorf genes are simultaneously transcribed in T2Bo strain B. bovis merozoites obtained from deep brain tissue of an acutely infected animal. SMORF peptide-specific antiserum bound in immunoblots to multiple proteins with a range of sizes predicted by smorf genes, confirming translation of smorf gene products from these transcripts. These results indicate that the smorf multigene family is larger than previously described and demonstrate that smorf genes are expressed and are undergoing variation, both within strains and in a lineage-specific pattern independent of strain specificity. The function of these novel proteins is unknown.