Both Coinfection and Superinfection Drive Complex Anaplasma marginale Strain Structure in a Natural Transmission Setting.

Vector-borne pathogens commonly establish multistrain infections, also called complex infections. How complex infections are established, either before or after the development of an adaptive immune response, termed coinfection or superinfection, respectively, has broad implications for the maintenance of genetic diversity, pathogen phenotype, epidemiology, and disease control strategies. Anaplasma marginale, a genetically diverse, obligate, intracellular, tick-borne bacterial pathogen of cattle, commonly establishes complex infections, particularly in regions with high transmission rates. Both coinfection and superinfection can be established experimentally; however, it is unknown how complex infections develop in a natural transmission setting. To address this question, we introduced naive animals into a herd in southern Ghana with a high infection prevalence and high transmission pressure and tracked the strain acquisition of A. marginale through time using multilocus sequence typing. As expected, the genetic diversity among strains was high, and 97% of animals in the herd harbored multiple strains. All the introduced naive animals became infected, and three to four strains were typically detected in an individual animal prior to seroconversion, while one to two new strains were detected in an individual animal following seroconversion. On average, the number of strains acquired via superinfection was 16% lower than the number acquired via coinfection. Thus, while complex infections develop via both coinfection and superinfection, coinfection predominates in this setting. These findings have broad implications for the development of control strategies in high-transmission settings.

Mycoplasma ovipneumoniae in Wildlife Species beyond Subfamily Caprinae.

Elucidating the emergence of Mycoplasma ovipneumoniae-associated respiratory disease in ruminants requires identification of the pathogen host range. This bacterium was thought to be host restricted to subfamily Caprinae, but we describe its identification in healthy moose, caribou, and mule deer and diseased mule and white-tailed deer, all species in subfamily Capreolinae.

Anaplasma marginale superinfection attributable to pathogen strains with distinct genomic backgrounds.

Strain superinfection occurs when a second pathogen strain infects a host already infected with a primary strain. The selective pressures that drive strain divergence, which underlies superinfection, and allow penetration of a new strain into a host population are critical knowledge gaps relevant to shifts in infectious disease epidemiology. In regions of endemicity with a high prevalence of infection, broad population immunity develops against Anaplasma marginale, a highly antigenically variant rickettsial pathogen, and creates strong selective pressure for emergence of and superinfection with strains that differ in their Msp2 variant repertoires. The strains may emerge either by msp2 locus duplication and allelic divergence on an existing genomic background or by introduction of a strain with a different msp2 allelic repertoire on a distinct genomic background. To answer this question, we developed a multilocus typing assay based on high-throughput sequencing of non-msp2 target loci to distinguish among strains with different genomic backgrounds. The technical error level was statistically defined based on the percentage of perfect sequence matches of clones of each target locus and validated using experimental single strains and strain pairs. Testing of A. marginale-positive samples from tropical regions where A. marginale infection is endemic identified individual infections that contained unique alleles for all five targeted loci. The data revealed a highly significant difference in the number of strains per animal in the tropical regions compared to infections in temperate regions and strongly supported the hypothesis that transmission of genomically distinct A. marginale strains predominates in high-prevalence areas of endemicity.

Coding complete genome of a strain of ovine herpesvirus-2 associated with a clinical case of malignant catarrhal fever in a domestic lamb.

Ovine herpesvirus-2 causes sheep-associated malignant catarrhal fever, a fatal disease of ruminants and pigs. The virus is carried by sheep, and infection is typically subclinical. Here, we report the coding complete genome sequence of a strain of OvHV-2 obtained from a clinically affected domestic lamb.

Identification of multilocus genetic heterogeneity in Anaplasma marginale subsp. centrale and its restriction following tick-borne transmission.

Anaplasma marginale subsp. centrale was the first vaccine used to protect against a rickettsial disease and is still in widespread use a century later. As its use preceded development of either cryopreservation or cell culture, the vaccine strain was maintained for decades by sequential passage among donor animals, excluding the natural tick-borne transmission cycle that provides a selective pressure or population "bottleneck." We demonstrated that the vaccine strain is genetically heterogeneous at 46 chromosomal loci and that heterogeneity was maintained upon inoculation into recipient animals. The number of variants per site ranged from 2 to 11 with a mean of 2.8/locus and a mode and median of 2/locus; variants included single-nucleotide polymorphisms, insertions/deletions, polynucleotide tracts, and different numbers of perfect repeats. The genetic heterogeneity is highly unlikely to be a result of strain contamination based on analysis using a panel of eight gene markers with a high power for strain discrimination. In contrast, heterogeneity appears to be a result of genetic drift in the absence of the restriction of tick passage. Heterogeneity could be reduced following tick passage, and the reduced heterogeneity could be maintained in sequential intravenous and tick-borne passages. The reduction in vaccine strain heterogeneity following tick passage did not confer an enhanced transmission phenotype, indicating that a stochastically determined population bottleneck was likely responsible as opposed to a positive selective pressure. These findings demonstrate the plasticity of an otherwise highly constrained genome and highlight the role of natural transmission cycles in shaping and maintaining the bacterial genome.

A U.S. Isolate of Theileria orientalis Ikeda Is Not Transstadially Transmitted to Cattle by Rhipicephalus microplus.

Theileria orientalis Ikeda has caused an epidemic of bovine anemia and abortion across several U.S. states. This apicomplexan hemoparasite is transmitted by Haemaphysalis longicornis ticks; however, it is unknown if other North American ticks are competent vectors. Since the disease movement is largely determined by the host tick range(s), the prediction of the T. orientalis spread among U.S. cattle populations requires determination of additional competent tick vectors. Although Rhipicephalus microplus has mostly been eradicated from the U.S., outbreaks in populations occur frequently, and the U.S. remains at risk for reintroduction. Since R. microplus is a vector of Theileria equi and T. orientalis DNA has been detected in R. microplus, the goal of this study was to determine whether R. microplus is a competent vector of T. orientalis. Larval R. microplus were applied to a splenectomized, T. orientalis Ikeda-infected calf for parasite acquisition, removed as molted adults, and applied to two T. orientalis naïve, splenectomized calves for transmission. After 60 days, the naïve calves remained negative for T. orientalis by PCR and cytology. Additionally, T. orientalis was not detected in the salivary glands or larval progeny of acquisition-fed adults. These data suggest that R. microplus is not a competent vector of the U.S. T. orientalis Ikeda isolate.

Comparative genomic analysis and phylogenetic position of Theileria equi.

BACKGROUND: Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites. RESULTS: The known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp. CONCLUSIONS: The EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.

LABORATORY CONCORDANCE STUDY FOR THE MOLECULAR DETECTION OF MYCOPLASMA OVIPNEUMONIAE.

As part of a respiratory pathogen survey of Alaska wildlife, we conducted a concordance study to assess Mycoplasma ovipneumoniae detection among three different PCR assays using a total of 346 nasal swabs sampled from four species (Dall's sheep, Ovis dalli dalli; mountain goats, Oreamnos americanus; caribou, Rangifer tarandus granti; and moose, Alces alces gigas), and two taxonomic subfamilies (Bovidae subfamily Caprinae and Cervidae subfamily Capreolinae). A federal research laboratory performed two PCR assays (LM40 and intergenic spacer region [IGS]), and a state diagnostic laboratory performed the third (universal Mycoplasma [UM]). Overall concordance was good, ranging from 93% to 99%, which was probably a result of low detection rate of M. ovipneumoniae. Due to differences in positive agreement, the quality of concordance between LM40 and both IGS and UM was considered fair. However, the quality of concordance between IGS and UM was excellent. All three PCR methods detected M. ovipneumoniae in a non-Caprinae species (caribou), and the LM40-PCR assay also detected M. ovipneumoniae in additional Caprinae species. The LM40-PCR assay detected M. ovipneumoniae in a larger number of samples than did the other two assays (IGS, UM). Because of potential differences in detection rates, it is critical to consider test parameters when evaluating a host population for the presence of M. ovipneumoniae.

Development of a real-time PCR assay for detection and differentiation of Mycoplasma ovipneumoniae and a novel respiratory-associated Mycoplasma species in domestic sheep and goats.

A novel respiratory-associated Mycoplasma species (M. sp. nov.) of unknown clinical significance was recently identified that causes false positive results with multiple published PCR methods reported to specifically detect Mycoplasma ovipneumonaie, a well-known respiratory pathogen in small ruminants. This necessitates our objective to develop a real-time PCR (qPCR) assay for improved specificity and sensitivity, and more rapid detection and differentiation of M. ovipneumoniae and the M. sp. nov. in domestic sheep (DS) and domestic goat (DG) samples, as compared to a conventional PCR and sequencing (cPCR-seq) assay. Primers and probes were designed based on available M. ovipneumoniae 16S rRNA gene sequences in the GenBank database, and partial 16S rRNA gene sequences provided by the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) for M. ovipneumoniae and M. sp. nov. USDA-ARS provided DS (n = 153) and DG (n = 194) nasal swab nucleic acid that previously tested positive for either M. ovipneumoniae (n = 117) or M. sp. nov. (n = 138), or negative for both targets (n = 92) by cPCR-seq. A host 18S rRNA gene was included as an internal control to monitor for the failure of nucleic acid extraction and possible PCR inhibition. For samples positive by cPCR-seq, qPCR agreement was 88.0% (103/117; κ = 0.81) and 89.9% (124/138; κ = 0.84) for M. ovipneumoniae and M. sp. nov., respectively; 12 of 255 (4.7%) cPCR-seq positive samples were qPCR positive for both targets. Of samples negative by cPCR for both mycoplasmas, qPCR detected M. ovipneumoniae and M. sp. nov. in 6.5% (6/92) and 4.3% (4/92), respectively. Samples with discordant results between the cPCR and sequencing assay and the new qPCR were analyzed by target sequencing; successfully sequenced samples had identity matches that confirmed the qPCR result. The increased target specificity of this qPCR is predicted to increase testing accuracy as compared to other published assays.

Draft Genome Sequence of a Novel Mycoplasma Species Identified from the Respiratory Tract of an Alaska Moose (Alces alces gigas).

The mycoplasmas represent a large and diverse group of bacteria, many of which are pathogens of humans and animals. Here, we describe a draft genome sequence of a novel Mycoplasma species. This novel Mycoplasma species has potential to cause false-positive PCR results for Mycoplasma ovipneumoniae, a respiratory-associated pathogen of ruminants.

Differential pulmonary immunopathology of domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) with Mycoplasma ovipneumoniae infection: A retrospective study.

Mycoplasma ovipneumoniae is a respiratory pathogen that impacts domestic sheep (Ovis aries; DS) and bighorn sheep (Ovis canadensis; BHS). BHS are reported to be more susceptible than DS to developing polymicrobial pneumonia associated with M. ovipneumoniae infection. Using formalin-fixed paraffin-embedded tissues, we performed a retrospective study investigating the pulmonary immune response of DS and BHS to M. ovipneumoniae infection. M. ovipneumoniae infected DS exhibited a more robust and well-organized BALT formation as compared to BHS. Digital analysis of immunohistochemical chromogen deposition in lung tissue was used to quantitate T cell marker CD3, B cell markers CD20 and CD79a, macrophage markers CD163 and Iba1, and cytokine IL-17. A significant interaction of species and infection status was identified for CD3, CD163, and IL-17. BHS had a greater increase in bronchiolar CD3 and bronchiolar and alveolar CD163 with infection, as compared to DS. BHS had an increase in bronchiolar associated lymph tissue (BALT) and alveolar IL-17 with infection, while these remained similar in DS regardless of infection status. IL-17 in respiratory epithelium of bronchi and bronchioles comparatively decreased in DS and increased in BHS with infection. These data begin to define the interspecies differential immune response to pulmonary M. ovipneumoniae infection in DS and BHS and provide the first investigations of respiratory epithelium-associated IL-17 in ovine.

Genes involved in immune, gene translation and chromatin organization pathways associated with Mycoplasma ovipneumoniae presence in nasal secretions of domestic sheep.

Mycoplasma ovipneumoniae contributes to polymicrobial pneumonia in domestic sheep. Elucidation of host genetic influences of M. ovipneumoniae nasal detection has the potential to reduce the incidence of polymicrobial pneumonia in sheep through implementation of selective breeding strategies. Nasal mucosal secretions were collected from 647 sheep from a large US sheep flock. Ewes of three breeds (Polypay n = 222, Rambouillet n = 321, and Suffolk n = 104) ranging in age from one to seven years, were sampled at three different times in the production cycle (February, April, and September/October) over four years (2015 to 2018). The presence and DNA copy number of M. ovipneumoniae was determined using a newly developed species-specific qPCR. Breed (P<0.001), age (P<0.024), sampling time (P<0.001), and year (P<0.001) of collection affected log10 transformed M. ovipneumoniae DNA copy number, where Rambouillet had the lowest (P<0.0001) compared with both Polypay and Suffolk demonstrating a possible genetic component to detection. Samples from yearlings, April, and 2018 had the highest (P<0.046) detected DNA copy number mean. Sheep genomic DNA was genotyped with the Illumina OvineHD BeadChip. Principal component analysis identified most of the variation in the dataset was associated with breed. Therefore, genome wide association analysis was conducted with a mixed model (EMMAX), with principal components 1 to 6 as fixed and a kinship matrix as random effects. Genome-wide significant (P<9x10-8) SNPs were identified on chromosomes 6 and 7 in the all-breed analysis. Individual breed analysis had genome-wide significant (P<9x10-8) SNPs on chromosomes 3, 4, 7, 9, 10, 15, 17, and 22. Annotated genes near these SNPs are part of immune (ANAPC7, CUL5, TMEM229B, PTPN13), gene translation (PIWIL4), and chromatin organization (KDM2B) pathways. Immune genes are expected to have increased expression when leukocytes encounter M. ovipneumoniae which would lead to chromatin reorganization. Work is underway to narrow the range of these associated regions to identify the underlying causal mutations.

A U.S. isolate of Theileria orientalis, Ikeda genotype, is transmitted to cattle by the invasive Asian longhorned tick, Haemaphysalis longicornis.

BACKGROUND: Theileria orientalis is a tick-borne hemoparasite that causes anemia, ill thrift, and death in cattle globally. The Ikeda strain of T. orientalis is more virulent than other strains, leading to severe clinical signs and death of up to 5% of affected animals. Within the Asia-Pacific region, where it affects 25% of Australian cattle, T. orientalis Ikeda has a significant economic impact on the cattle industry. In 2017, T. orientalis Ikeda was detected in a cattle herd in Albermarle County, Virginia, United States. Months earlier, the U.S. was alerted to the invasion of the Asian longhorned tick, Haemaphysalis longicornis, throughout the eastern U.S. Abundant H. longicornis ticks were identified on cattle in the T. orientalis-affected herd in VA, and a subset of ticks from the environment were PCR-positive for T. orientalis Ikeda. A strain of T. orientalis from a previous U.S. outbreak was not transmissible by H. longicornis; however, H. longicornis is the primary tick vector of T. orientalis Ikeda in other regions of the world. Thus, the objective of this study was to determine whether invasive H. longicornis ticks in the U.S. are competent vectors of T. orientalis Ikeda. METHODS: Nymphal H. longicornis ticks were fed on a splenectomized calf infected with the VA-U.S.-T. orientalis Ikeda strain. After molting, a subset of adult ticks from this cohort were dissected, and salivary glands assayed for T. orientalis Ikeda via qPCR. The remaining adult ticks from the group were allowed to feed on three calves. Calves were subsequently monitored for T. orientalis Ikeda infection via blood smear cytology and PCR. RESULTS: After acquisition feeding on a VA-U.S.-T. orientalis Ikeda-infected calf as nymphs, a subset of molted adult tick salivary glands tested positive by qPCR for T. orientalis Ikeda. Adult ticks from the same cohort successfully transmitted T. orientalis Ikeda to 3/3 naïve calves, each of which developed parasitemia reaching 0.4-0.9%. CONCLUSIONS: Our findings demonstrate that U.S. H. longicornis ticks are competent vectors of the VA-U.S.-T. orientalis Ikeda strain. This data provides important information for the U.S. cattle industry regarding the potential spread of this parasite and the necessity of enhanced surveillance and control measures.

Comparative analysis of gene expression between Babesia bovis blood stages and kinetes allowed by improved genome annotation.

Throughout their life cycle, Babesia parasites alternate between a mammalian host, where they cause babesiosis, and the tick vector. Transition between hosts results in distinct environmental signals that influence patterns of gene expression, consistent with the morphological and functional changes operating in the parasites during their life stages. In addition, comparing differential patterns of gene expression among mammalian and tick parasite stages can provide clues for developing improved methods of control. Hereby, we upgraded the genome assembly of Babesia bovis, a bovine hemoparasite, closing a 139 kbp gap, and used RNA-Seq datasets derived from mammalian blood and tick kinete stages to update the genome annotation. Of the originally annotated genes, 1,254 required structural changes, and 326 new genes were identified, leading to a different predicted proteome compared to the original annotation. Next, the RNA-Seq data was used to identify B. bovis genes that were differentially expressed in the vertebrate and arthropod hosts. In blood stages, 28% of the genes were upregulated up to 300 fold, whereas 26% of the genes in kinetes, a tick stage, were upregulated up to >19,000 fold. We thus discovered differentially expressed genes that may play key biological roles and serve as suitable targets for the development of vaccines to control bovine babesiosis.

Complete genome sequence of Anaplasma marginale subsp. centrale.

Anaplasma marginale subsp. centrale is a naturally attenuated subtype that has been used as a vaccine for a century. We sequenced the genome of this organism and compared it to those of virulent senso stricto A. marginale strains. The comparison markedly narrows the number of outer membrane protein candidates for development of a safer inactivated vaccine and provides insight into the diversity among strains of senso lato A. marginale.

Genome sequences of Mannheimia haemolytica serotype A2: ovine and bovine isolates.

This report describes the genome sequences of Mannheimia haemolytica serotype A2 isolated from pneumonic lungs of two different ruminant species, one from Ovis aries, designated ovine (O), and the other from Bos taurus, designated bovine (B).

blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids.

Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from Escherichia coli (cattle) and Salmonella enterica serovar Newport (human) and that carry the cephamycinase gene blaCMY-2. These large plasmids (148 to 166 kbp) share extensive sequence identity and synteny. The most divergent plasmid, peH4H, has lost several conjugation-related genes and has gained a kanamycin resistance region. Two of the plasmids (pAM04528 and peH4H) harbor two copies of blaCMY-2, while the third plasmid (pAR060302) harbors a single copy of the gene. The majority of single-nucleotide polymorphisms comprise nonsynonymous mutations in floR. A comparative analysis of these plasmids with five other published IncA/C plasmids showed that the blaCMY-2 plasmids from E. coli and S. enterica are genetically distinct from those originating from Yersinia pestis and Photobacterium damselae and distal to one originating from Yersinia ruckeri. While the overall similarity of these plasmids supports the likelihood of recent movements among E. coli and S. enterica hosts, their greater divergence from Y. pestis or Y. ruckeri suggests less recent plasmid transfer among these pathogen groups.

Genome-Wide Histone Modifications and CTCF Enrichment Predict Gene Expression in Sheep Macrophages.

Alveolar macrophages function in innate and adaptive immunity, wound healing, and homeostasis in the lungs dependent on tissue-specific gene expression under epigenetic regulation. The functional diversity of tissue resident macrophages, despite their common myeloid lineage, highlights the need to study tissue-specific regulatory elements that control gene expression. Increasing evidence supports the hypothesis that subtle genetic changes alter sheep macrophage response to important production pathogens and zoonoses, for example, viruses like small ruminant lentiviruses and bacteria like Coxiella burnetii. Annotation of transcriptional regulatory elements will aid researchers in identifying genetic mutations of immunological consequence. Here we report the first genome-wide survey of regulatory elements in any sheep immune cell, utilizing alveolar macrophages. We assayed histone modifications and CTCF enrichment by chromatin immunoprecipitation with deep sequencing (ChIP-seq) in two sheep to determine cis-regulatory DNA elements and chromatin domain boundaries that control immunity-related gene expression. Histone modifications included H3K4me3 (denoting active promoters), H3K27ac (active enhancers), H3K4me1 (primed and distal enhancers), and H3K27me3 (broad silencers). In total, we identified 248,674 reproducible regulatory elements, which allowed assignment of putative biological function in macrophages to 12% of the sheep genome. Data exceeded the FAANG and ENCODE standards of 20 million and 45 million useable fragments for narrow and broad marks, respectively. Active elements showed consensus with RNA-seq data and were predictive of gene expression in alveolar macrophages from the publicly available Sheep Gene Expression Atlas. Silencer elements were not enriched for expressed genes, but rather for repressed developmental genes. CTCF enrichment enabled identification of 11,000 chromatin domains with mean size of 258 kb. To our knowledge, this is the first report to use immunoprecipitated CTCF to determine putative topological domains in sheep immune cells. Furthermore, these data will empower phenotype-associated mutation discovery since most causal variants are within regulatory elements.

Transcriptome dataset of Babesia bovis life stages within vertebrate and invertebrate hosts.

Babesia bovis is a hemoprotozoan parasite of cattle that has a complex life cycle within vertebrate and invertebrate hosts. In the mammalian host, B. bovis undergoes asexual reproduction while in the tick midgut, gametes are induced, fuse, and form zygotes. The zygote infects tick gut epithelial cells and transform into kinetes that are released into the hemolymph and invade other tick tissues such as the ovaries, resulting in transovarial transmission to tick offspring. To compare gene regulation between different B. bovis life stages, we collected parasites infecting bovine erythrocytes and tick hemolymph. Total RNA samples were isolated, and multiplexed libraries sequenced using paired-end 100 cycle reads of a HiSeq 2500. The data was normalized using the TMM method and analysed for significant differential expression using the generalized linear model likelihood ratio test (GLM LRT) in edgeR. To validate our datasets, ten genes were selected using NormFinder. Genes that had no significant fold change between the blood and tick stages in the RNA-Seq datasets were tested by quantitative PCR to determine their suitability as "housekeeping" genes. The normalized RNA-Seq data revealed genes upregulated during infection of the mammalian host or tick vector and six upregulated genes were validated by quantitative PCR. These datasets can help identify useful targets for controlling bovine babesiosis.