Bovine Host Research Articles

Differentially expressed genes in response to amitraz treatment suggests a proposed model of resistance to amitraz in R. decoloratus ticks

The widespread geographical distribution of Rhipicephalus decoloratus in southern Africa and its ability to transmit the pathogens causing redwater, gallsickness and spirochaetosis in cattle makes this hematophagous ectoparasite of economic importance. In South Africa, the most commonly used chemical acaricides to control tick populations are pyrethroids and amitraz. The current amitraz resistance mechanism described in R. microplus, from South Africa and Australia, involves mutations in the octopamine receptor, but it is unlikely that this will be the only contributing factor to mediate resistance. Therefore, in this study we aimed to gain insight into the more complex mechanism(s) underlying amitraz resistance in R. decoloratus using RNA-sequencing. Differentially expressed genes (DEGs) were identified when comparing amitraz susceptible and resistant ticks in the presence of amitraz while fed on bovine hosts. The most significant DEGs were further analysed using several annotation tools. The predicted annotations from these genes, as well as KEGG pathways potentially point towards a relationship between the α-adrenergic-like octopamine receptor and ionotropic glutamate receptors in establishing amitraz resistance. All genes with KEGG pathway annotations were further validated using RT-qPCR across all life stages of the tick. In susceptible ticks, the proposed model is that in the presence of amitraz, there is inhibition of Ca2+ entry into cells and subsequent membrane hyperpolarization which prevents the release of neurotransmitters. In resistant ticks, we hypothesize that this is overcome by ionotropic glutamate receptors (NMDA and AMPA) to enhance synaptic transmission and plasticity in the presence of neurosteroids. Activation of NMDA receptors initiates long term potentiation (LTP) which may allow the ticks to respond more rapidly and with less stimulus when exposed to amitraz in future. Overactivation of the NMDA receptor and excitotoxicity is attenuated by the estrone, NAD+ and ATP hydrolysing enzymes. This proposed pathway paves the way to future studies on understanding amitraz resistance and should be validated using in vivo activity assays (through the use of inhibitors or antagonists) in combination with metabolome analyses.

Selected reaction monitoring mass spectrometry of mastitis milk reveals pathogen-specific regulation of bovine host response proteins

Mastitis is a major challenge to bovine health. The detection of sensitive markers for mastitis in dairy herds is of great demand. Suitable biomarkers should be measurable in milk and should report pathogen-specific changes at an early stage to support earlier diagnosis and more efficient treatment. However, the identification of sensitive biomarkers in milk has remained a challenge, in part due to their relatively low concentration in milk. In the present study, we used a selected reaction monitoring (SRM) mass spectrometry approach, which allowed the absolute quantitation of 13 host response proteins in milk for the first time. These proteins were measured over a 54-h period upon an in vivo challenge with cell wall components from either gram-negative (lipopolysaccharide from Escherichia coli; LPS) or gram-positive bacteria (peptidoglycan from Staphylococcus aureus; PGN). Whereas our data clearly demonstrate that all challenged animals have consistent upregulation of innate immune response proteins after both LPS and PGN challenge, the data also reveal clearly that LPS challenge unleashes faster and shows a more intense host response compared with PGN challenge. Biomarker candidates that may distinguish between gram-negative and gram-positive bacteria include α-2 macroglobulin, α-1 antitrypsin, haptoglobin, serum amyloid A3, cluster of differentiation 14, calgranulin B, cathepsin C, vanin-1, galectin 1, galectin 3, and IL-8. Our approach can support further studies of large cohorts of animals with natural occurring mastitis, to validate the relevance of these suggested biomarkers in dairy production.

Phylogenetic signature of lateral exchange of genes for antibiotic production and resistance among bacteria highlights a pattern of global transmission of pathogens between humans and livestock

The exchange of bacterial virulence factors driven by lateral gene transfer (LGT) can help indicate possible bacterial transmission among different hosts. Specifically, overlaying the phylogenetic signal of LGT among bacteria onto the distribution of respective isolation sources (hosts) can indicate patterns of transmission among these hosts. Here, we apply this approach towards a better understanding of patterns of bacterial transmission between humans and livestock. We utilize comparative genomics to trace patterns of LGT for an 11-gene operon responsible for the production of the antibiotic nisin and infer transmission of bacteria among respective host species. A total of 147 bacterial genomes obtained from NCBI were determined to contain the complete operon. Isolated from human, porcine and bovine hosts, these genomes represented six Streptococcus and one Staphylococcus species. Phylogenetic analyses of the operon sequences revealed a signature of frequent and recent lateral gene transfer that indicated extensive bacterial transmission between humans and pigs. For 11 isolates, we detected a Tn916-like transposon inserted into the operon. The transposon contained the tetM gene (tetracycline resistance) and additional phylogenetic analyses indicated transmission among human and animal hosts. The bacteria possessing the nisin operon and transposon were isolated from hosts distributed globally. These findings possibly reflect both the globalization of the food industry and an increasingly mobile and expanding human population. In addition to concerns regarding zoonosis, these findings also highlight the potential threat to livestock worldwide due to reverse zoonosis.

Immunobiotics for the Bovine Host: Their Interaction with Intestinal Epithelial Cells and Their Effect on Antiviral Immunity.

The scientific community has reported several cases of microbes that exhibit elevated rates of antibiotic resistance in different regions of the planet. Due to this emergence of antimicrobial resistant microorganisms, the use of antibiotics as promoters of livestock animals’ growth is being banned in most countries around the world. One of the challenges of agricultural immunology therefore is to find alternatives by modulating the immune system of animals in drug-independent safe food production systems. In this regard, in an effort to supplant antibiotics from bovine feeds, several alternatives were proposed including the use of immunomodulatory probiotics (immunobiotics). The purpose of this review is to provide an update of the status of the modulation of intestinal antiviral innate immunity of the bovine host by immunobiotics, and the beneficial impact of immunobiotics on viral infections, focused on intestinal epithelial cells (IECs). The results of our group, which demonstrate the capacity of immunobiotic strains to beneficially modulate Toll-like receptor 3-triggered immune responses in bovine IECs and improve the resistance to viral infections, are highlighted. This review provides comprehensive information on the innate immune response of bovine IECs against virus, which can be further investigated for the development of strategies aimed to improve defenses in the bovine host.

Ecological preferences and seasonal dynamics of ticks (Acari: Ixodidae) on and off bovine hosts in the Eastern Cape Province, South Africa.

A total of 31,425 ticks were collected from 10 cattle and also from six drag-samples during the 12-month study period. Adult ticks were removed from the right hand side of each animal and all instars of ticks were placed in containers filled with 70% ethanol. Based on morphological traits, 10 tick species were identified: Rhipicephalus (Boophilus) decoloratus (32.5%), R. evertsi evertsi (18.8%), R. appendiculatus (17.3%), Amblyomma hebraeum (16.3%), R. simus (7.7%), Ixodes pilosus (3.8%), Hyalomma rufipes (3.5%), R. follis (0.08%), Haemaphysalis elliptica (0.04%), H. silacea (0.02%). The southern African yellow dog tick, H. elliptica, was only found on vegetation. The agro-ecological zones differ significantly in tick species and their distribution. The A. hebraeum and R. evertsi evertsi counts were higher in Kowie Thicket (KT) during summer season (2.05±0.01 and 1.00±0.09, respectively) compared to Bedford Dry Grassland (BDG) and Bhisho Thornveld (BT) veld types. In all vegetation types, R. appendiculatus had higher counts in KT in spring (0.91±0.08), summer (0.78±0.08) and winter (0.78±0.08). Rhipicephalus (Boophilus) decoloratus was more frequent in the BT (1.78±0.11) during the summer season. BDG had lower tick infestation with R. evertsi evertsi being the most frequent species in summer. No H. rufipes was collected in the KT. Of epidemiological interest, R. (B.) microplus was absent in the study area which needs further investigation. Within the context of this study, we found agro-ecological differences and seasonal variations to have influence on tick species distribution.

Bovine Immune Factors Underlying Tick Resistance: Integration and Future Directions.

The mechanisms underlying tick resistance within and between cattle breeds have been studied for decades. Several previous papers on bovine immune parameters contributing to tick resistance discussed findings across DNA, RNA, protein, cellular, and tissue levels. However, the differences between bovine host species, tick species and the experimental layouts were not always taken into account. This review aims to (a) give a comprehensive summary of studies investigating immune marker differences between cattle breeds with varying degrees of tick resistance, and (b) to integrate key findings and suggest hypotheses on likely immune-regulated pathways driving resistance. Experimental issues, which may have skewed conclusions, are highlighted. In future, improved experimental strategies will enable more focused studies to identify and integrate immune markers and/or pathways. Most conclusive thus far is the involvement of histamine, granulocytes and their associated pathways in the tick-resistance mechanism. Interestingly, different immune markers might be involved in the mechanisms within a single host breed in contrast to between breeds. Also, differences are evident at each tick life stage, limiting the level to which datasets can be compared. Future studies to further elucidate immune molecule dynamics across the entire tick life cycle and in-depth investigation of promising markers and pathways on both molecular and cellular level are in dire need to obtain a scientifically sound hypothesis on the drivers of tick resistance.

Cattle Tick Rhipicephalus microplus-Host Interface: A Review of Resistant and Susceptible Host Responses.

Ticks are able to transmit tick-borne infectious agents to vertebrate hosts which cause major constraints to public and livestock health. The costs associated with mortality, relapse, treatments, and decreased production yields are economically significant. Ticks adapted to a hematophagous existence after the vertebrate hemostatic system evolved into a multi-layered defense system against foreign invasion (pathogens and ectoparasites), blood loss, and immune responses. Subsequently, ticks evolved by developing an ability to suppress the vertebrate host immune system with a devastating impact particularly for exotic and crossbred cattle. Host genetics defines the immune responsiveness against ticks and tick-borne pathogens. To gain an insight into the naturally acquired resistant and susceptible cattle breed against ticks, studies have been conducted comparing the incidence of tick infestation on bovine hosts from divergent genetic backgrounds. It is well-documented that purebred and crossbred Bos taurus indicus cattle are more resistant to ticks and tick-borne pathogens compared to purebred European Bos taurus taurus cattle. Genetic studies identifying Quantitative Trait Loci markers using microsatellites and SNPs have been inconsistent with very low percentages relating phenotypic variation with tick infestation. Several skin gene expression and immunological studies have been undertaken using different breeds, different samples (peripheral blood, skin with tick feeding), infestation protocols and geographic environments. Susceptible breeds were commonly found to be associated with the increased expression of toll like receptors, MHC Class II, calcium binding proteins, and complement factors with an increased presence of neutrophils in the skin following tick feeding. Resistant breeds had higher levels of T cells present in the skin prior to tick infestation and thus seem to respond to ticks more efficiently. The skin of resistant breeds also contained higher numbers of eosinophils, mast cells and basophils with up-regulated proteases, cathepsins, keratins, collagens and extracellular matrix proteins in response to feeding ticks. Here we review immunological and molecular determinants that explore the cattle tick Rhipicephalus microplus-host resistance phenomenon as well as contemplating new insights and future directions to study tick resistance and susceptibility, in order to facilitate interventions for tick control.

Population structure and virulence gene profiles of Streptococcus agalactiae collected from different hosts worldwide.

Streptococcus agalactiae is a leading cause of morbidity and mortality among neonates and causes severe infections in pregnant women and nonpregnant predisposed adults, in addition to various animal species worldwide. Still, information on the population structure of S. agalactiae and the geographical distribution of different clones is limited. Further data are urgently needed to identify particularly successful clones and obtain insights into possible routes of transmission within one host species and across species borders. We aimed to determine the population structure and virulence gene profiles of S. agalactiae strains from a diverse set of sources and geographical origins. To this end, 373S. agalactiae isolates obtained from humans and animals from five different continents were typed by DNA microarray profiling. A total of 242 different S. agalactiae strains were identified and further analyzed. Particularly successful clonal lineages, hybridization patterns, and strains were identified that were spread across different continents and/or were present in more than one host species. In particular, several strains were detected in both humans and cattle, and several canine strains were also detected in samples from human, bovine, and porcine hosts. The findings of our study suggest that although S. agalactiae is well adapted to various hosts including humans, cattle, dogs, rodents, and fish, interspecies transmission is possible and occurs between humans and cows, dogs, and rabbits. The virulence and resistance gene profiles presented enable new insights into interspecies transmission and make a crucial contribution to the identification of suitable targets for therapeutic agents and vaccines.

Distinct Campylobacter fetus lineages adapted as livestock pathogens and human pathobionts in the intestinal microbiota

Campylobacter fetus is a venereal pathogen of cattle and sheep, and an opportunistic human pathogen. It is often assumed that C. fetus infection occurs in humans as a zoonosis through food chain transmission. Here we show that mammalian C. fetus consists of distinct evolutionary lineages, primarily associated with either human or bovine hosts. We use whole-genome phylogenetics on 182 strains from 17 countries to provide evidence that C. fetus may have originated in humans around 10,500 years ago and may have “jumped” into cattle during the livestock domestication period. We detect C. fetus genomes in 8% of healthy human fecal metagenomes, where the human-associated lineages are the dominant type (78%). Thus, our work suggests that C. fetus is an unappreciated human intestinal pathobiont likely spread by human to human transmission. This genome-based evolutionary framework will facilitate C. fetus epidemiology research and the development of improved molecular diagnostics and prevention schemes for this neglected pathogen.

Genomic comparison of bovine papillomavirus 1 isolates from bovine, equine and asinine lesional tissue samples

Several attempts have been made to categorize equid- and bovid-specific bovine papillomavirus 1 (BPV1) isolates based on sequence tags. This study includes newly determined sequence information from 33 BPV1 isolates of equine, asinine and bovine origin and investigates sequence bias due to host species. Twenty of the viral genomes were sequenced over their entire length and a further thirteen were sequenced, including flanking sequences, at two specific sites, the LCR and the E5 ORF. Alignment and analyses of the sequences did not reveal statistically significant site differences between the sequences of bovine and equid origin. None of the proposed sites of divergence noted by other authors demonstrated significant species-specific characteristics. Our results suggest that BPV1 is shared between equine, asinine and bovine host species, and that viral transfer between bovines and equids is a repeated and ongoing phenomenon.

Ixodid Ticks (Arachnida: Acari) Prevalence Associated with Risk Factors in the Bovine Host in District Quetta, Baluchistan

Ixodid Ticks (Arachnida: Acari) Prevalence Associated with Risk Factors in the Bovine Host in District Quetta, Baluchistan

Draft genome sequences of two fluoroquinolone-resistant CTX-M-15-producing Escherichia coli ST90 (ST23 complex) isolated from a calf and a dairy cow in South America.

Farm animals have been recognised as important carriers and reservoirs of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli. The aim of this study was to report the draft genome sequences of two multidrug-resistant (MDR) CTX-M-15-producing E. coli strains (47VL and 13B) isolated from different bovine hosts (a calf and a dairy cow), housed separately in a commercial dairy farm in Brazil. Total genomic DNA of the E. coli isolates was sequenced using an Illumina MiSeq paired-end 300-bp sequencing platform. Sequence reads were de novo assembled using the A5-miseq pipeline and polishing assembly in Geneious v.R9. The NCBI Prokaryotic Genome Annotation Pipeline v.3.2 was used for genome annotation, whereas whole-genome sequences were analysed using bioinformatic tools from the Center of Genomic Epidemiology and EnteroBase. E. coli 47VL generated a total of 3238770 and E. coli 13B a total of 1422808 paired-end reads of ca. 190× and ca. 80×, respectively. The resistome revealed that both isolates carried resistance genes to aminoglycosides, β-lactams, macrolides, sulphonamides, trimethoprim and tetracycline. Comparative analyses revealed clonal relatedness. In fact, both isolates belonged to sequence type ST90 (clonal complex CC23) and phylogroup AxB1. To our knowledge, these are the first draft genome sequences of CTX-M-15-producing E. coli ST90 isolated from bovines in South America. These data can be used to elucidate genetic features that contribute to colonisation and adaptation of CTX-M-15-producing E. coli in dairy cattle.

Strong influence of livestock environments on the emergence and dissemination of distinct multidrug-resistant phenotypes among the population of non-typhoidal Salmonella.

The problem of emergence and dissemination of multidrug resistance, especially among Gram-negative bacteria, has reached alarming levels. This increases the need to develop surveillance methods that more effectively and accurately provide information about the emergence and spread of multidrug-resistant organisms. In this study, using a well-defined population of non-typhoidal Salmonella (NTS) isolates associated with avian, bovine and porcine hosts, we found that the livestock environments had a specific (P < 0.005) and profound (P < 0.005) effect on the evolution of multidrug-resistant phenotypes among population of NTS isolates. The MDR pattern containing penicillins, tetracyclines and macrolides and the evolving counterparts (i.e., penicillins, tetracyclines and macrolides + other antibiotic classes) were significantly (P < 0.005) associated with NTS isolates of porcine origin. Similarly, MDR patterns containing folate pathway inhibitors, macrolides and aminocyclitol or containing penicillins, cephalosporins, tetracyclines, phenicols and macrolides were significantly associated with avian (P < 0.005) and bovine (P < 0.005) NTS isolates, respectively. Furthermore, STRUCTURE, an evolutionary analysis, clearly showed that the host origin (i.e., livestock environment), and not the genetic background of different NTS serovars, was the most determinative factor for acquisition and spread of MDR phenotypes. In addition, we described a novel non-synonymous mutation, located outside of the QRDR at position 864 of GyrA, that was likely associated with fluoroquinolone resistance.

An in silico pan-genomic probe for the molecular traits behind Lactobacillus ruminis gut autochthony.

As an ecological niche, the mammalian intestine provides the ideal habitat for a variety of bacterial microorganisms. Purportedly, some commensal genera and species offer a beneficial mix of metabolic, protective, and structural processes that help sustain the natural digestive health of the host. Among these sort of gut inhabitants is the Gram-positive lactic acid bacterium Lactobacillus ruminis, a strict anaerobe with both pili and flagella on its cell surface, but also known for being autochthonous (indigenous) to the intestinal environment. Given that the molecular basis of gut autochthony for this species is largely unexplored and unknown, we undertook a study at the genome level to pinpoint some of the adaptive traits behind its colonization behavior. In our pan-genomic probe of L. ruminis, the genomes of nine different strains isolated from human, bovine, porcine, and equine host guts were compiled and compared for in silico analysis. For this, we conducted a geno-phenotypic assessment of protein-coding genes, with an emphasis on those products involved with cell-surface morphology and anaerobic fermentation and respiration. We also categorized and examined the core and accessory genes that define the L. ruminis species and its strains. Here, we made an attempt to identify those genes having ecologically relevant phenotypes that might support or bring about intestinal indigenousness.

Permissiveness of bovine epithelial cells from lung, intestine, placenta and udder for infection with Coxiella burnetii

Ruminants are the main source of human infections with the obligate intracellular bacterium Coxiella (C.) burnetii. Infected animals shed high numbers of C. burnetii by milk, feces, and birth products. In goats, shedding by the latter route coincides with C. burnetii replication in epithelial (trophoblast) cells of the placenta, which led us to hypothesize that epithelial cells are generally implicated in replication and shedding of C. burnetii. We therefore aimed at analyzing the interactions of C. burnetii with epithelial cells of the bovine host (1) at the entry site (lung epithelium) which govern host immune responses and (2) in epithelial cells of gut, udder and placenta decisive for the quantity of pathogen excretion. Epithelial cell lines [PS (udder), FKD-R 971 (small intestine), BCEC (maternal placenta), F3 (fetal placenta), BEL-26 (lung)] were inoculated with C. burnetii strains Nine Mile I (NMI) and NMII at different cultivation conditions. The cell lines exhibited different permissiveness for C. burnetii. While maintaining cell viability, udder cells allowed the highest replication rates with formation of large cell-filling Coxiella containing vacuoles. Intestinal cells showed an enhanced susceptibility to invasion but supported C. burnetii replication only at intermediate levels. Lung and placental cells also internalized the bacteria but in strikingly smaller numbers. In any of the epithelial cells, both Coxiella strains failed to trigger a substantial IL-1β, IL-6 and TNF-α response. Epithelial cells, with mammary epithelial cells in particular, may therefore serve as a niche for C. burnetii replication in vivo without alerting the host’s immune response.

Molecular Characterization of Staphylococcus aureus Isolated from Bovine Mastitis and Close Human Contacts in South African Dairy Herds: Genetic Diversity and Inter-Species Host Transmission.

Staphylococcus aureus is one of the most common etiological agents of contagious bovine mastitis worldwide. The purpose of this study was to genetically characterize a collection of S. aureus isolates (bovine = 146, human = 12) recovered from cases of bovine mastitis and nasal swabs of close human contacts in the dairy environment. Isolates were screened for a combination of clinically significant antimicrobial and virulence gene markers whilst the molecular epidemiology of these isolates and possible inter-species host transmission was investigated using a combination of genotyping techniques. None of the isolates under evaluation tested positive for methicillin or vancomycin resistance encoding genes. Twenty seven percent of the bovine S. aureus isolates tested positive for one or more of the pyrogenic toxin superantigen (PTSAg) genes with the sec and sell genes predominating. Comparatively, 83% of the human S. aureus isolates tested positive for one or more PTSAg genes with a greater variety of genes being detected. Genomic DNA macrorestriction followed by pulsed-field gel electrophoresis (PFGE) of the bovine isolates generated 58 electrophoretic patterns which grouped into 10 pulsotypes at an 80% similarity level. The majority of the bovine isolates, 93.2% (136/146), clustered into four major pulsotypes. Seven sequence types (ST) were identified among the representative bovine S. aureus isolates genotyped, including: ST8 (CC8), ST97 (CC97), ST351 (CC705), ST352 (CC97), ST508 (CC45), ST2992 (CC97) and a novel sequence type, ST3538 (CC97). Based on PFGE analysis, greater genetic diversity was observed among the human S. aureus isolates. Bovine and human isolates from three sampling sites clustered together and were genotypically indistinguishable. Two of the isolates, ST97 and ST352 belong to the common bovine lineage CC97, and their isolation from close human contacts suggests zoonotic transfer. In the context of this study, the third isolate, ST8 (CC8), is believed to be a human clone which has transferred to a dairy cow and has subsequently caused mastitis. The detection of indistinguishable S. aureus isolates from bovine and human hosts at three of the sampling sites is suggestive of bacterial transmission and supports the need for vigilant monitoring of staphylococcal populations at the human-animal interface.

Immune recognition of salivary proteins from the cattle tick Rhipicephalus microplus differs according to the genotype of the bovine host

BackgroundMales of the cattle tick Rhipicephalus microplus produce salivary immunoglobulin-binding proteins and allotypic variations in IgG are associated with tick loads in bovines. These findings indicate that antibody responses may be essential to control tick infestations. Infestation loads with cattle ticks are heritable: some breeds carry high loads of reproductively successful ticks, in others, few ticks feed and they reproduce inefficiently. Different patterns of humoral immunity against tick salivary proteins may explain these phenotypes.MethodsWe describe the profiles of humoral responses against tick salivary proteins elicited during repeated artificial infestations of bovines of a tick-resistant (Nelore) and a tick-susceptible (Holstein) breed. We measured serum levels of total IgG1, IgG2 and IgE immunoglobulins and of IgG1 and IgG2 antibodies specific for tick salivary proteins. With liquid chromatography followed by mass spectrometry we identified tick salivary proteins that were differentially recognized by serum antibodies from tick-resistant and tick-susceptible bovines in immunoblots of tick salivary proteins separated by two-dimensional electrophoresis.ResultsBaseline levels of total IgG1 and IgG2 were significantly higher in tick-susceptible Holsteins compared with resistant Nelores. Significant increases in levels of total IgG1, but not of IgG2 accompanied successive infestations in both breeds. Resistant Nelores presented with significantly higher levels of salivary-specific antibodies before and at the first challenge with tick larvae; however, by the third challenge, tick-susceptible Holsteins presented with significantly higher levels of IgG1 and IgG2 tick salivary protein-specific antibodies. Importantly, sera from tick-resistant Nelores reacted with 39 tick salivary proteins in immunoblots of salivary proteins separated in two dimensions by electrophoresis versus only 21 spots reacting with sera from tick-susceptible Holsteins.ConclusionsLevels of tick saliva-specific antibodies were not directly correlated with infestation phenotypes. However, in spite of receiving apparently lower amounts of tick saliva, tick-resistant bovines recognized more tick salivary proteins. These reactive salivary proteins are putatively involved in several functions of parasitism and blood-feeding. Our results indicate that neutralization by host antibodies of tick salivary proteins involved in parasitism is essential to control tick infestations.

Analysis of Stage-Specific Protein Expression during Babesia Bovis Development within Female Rhipicephalus Microplus.

Arthropod-borne protozoan pathogens have a complex life cycle that includes asexual reproduction of haploid stages in mammalian hosts and the development of diploid stages in invertebrate hosts. The ability of pathogens to invade, survive, and replicate within distinct cell types is required to maintain their life cycle. In this study, we describe a comparative proteomic analysis of a cattle pathogen, Babesia bovis, during its development within the mammalian and tick hosts with the goal of identifying cell-surface proteins expressed by B. bovis kinetes as potential targets for the development of a transmission blocking vaccine. To determine parasite tick-stage-specific cell-surface proteins, CyDye labeling was performed with B. bovis blood stages from the bovine host and kinetes from the tick vector. Cell-surface kinete-stage-specific proteins were identified using 2D difference in gel electrophoresis and analyzed by mass spectrometry. Ten proteins were identified as kinete-stage-specific, with orthologs found in closely related Apicomplexan pathogens. Transcriptional analysis revealed two genes were highly expressed by kinetes as compared with blood stages. Immunofluorescence using antibodies against the two proteins confirmed kinete-stage-specific expression. The identified cell-surface kinete proteins are potential candidates for the development of a B. bovis transmission blocking vaccine.

Immune and biochemical responses in skin differ between bovine hosts genetically susceptible and resistant to the cattle tick Rhipicephalus microplus

BackgroundTicks attach to and penetrate their hosts’ skin and inactivate multiple components of host responses in order to acquire a blood meal. Infestation loads with the cattle tick, Rhipicephalus microplus, are heritable: some breeds carry high loads of reproductively successful ticks, whereas in others, few ticks feed and reproduce efficiently.MethodsIn order to elucidate the mechanisms that result in the different outcomes of infestations with cattle ticks, we examined global gene expression and inflammation induced by tick bites in skins from one resistant and one susceptible breed of cattle that underwent primary infestations with larvae and nymphs of R. microplus. We also examined the expression profiles of genes encoding secreted tick proteins that mediate parasitism in larvae and nymphs feeding on these breeds.ResultsFunctional analyses of differentially expressed genes in the skin suggest that allergic contact-like dermatitis develops with ensuing production of IL-6, CXCL-8 and CCL-2 and is sustained by HMGB1, ISG15 and PKR, leading to expression of pro-inflammatory chemokines and cytokines that recruit granulocytes and T lymphocytes. Importantly, this response is delayed in susceptible hosts. Histopathological analyses of infested skins showed inflammatory reactions surrounding tick cement cones that enable attachment in both breeds, but in genetically tick-resistant bovines they destabilized the cone. The transcription data provided insights into tick-mediated activation of basophils, which have previously been shown to be a key to host resistance in model systems. Skin from tick-susceptible bovines expressed more transcripts encoding enzymes that detoxify tissues. Interestingly, these enzymes also produce volatile odoriferous compounds and, accordingly, skin rubbings from tick-susceptible bovines attracted significantly more tick larvae than rubbings from resistant hosts. Moreover, transcripts encoding secreted modulatory molecules by the tick were significantly more abundant in larval and in nymphal salivary glands from ticks feeding on susceptible bovines.ConclusionsCompared with tick-susceptible hosts, genes encoding enzymes producing volatile compounds exhibit significantly lower expression in resistant hosts, which may render them less attractive to larvae; resistant hosts expose ticks to an earlier inflammatory response, which in ticks is associated with significantly lower expression of genes encoding salivary proteins that suppress host immunity, inflammation and coagulation.

Inhibition of Antigen-Specific and Nonspecific Stimulation of Bovine T and B Cells by Lymphostatin from Attaching and Effacing Escherichia coli.

ABSTRACTEnterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are enteric bacterial pathogens of worldwide importance. Most EPEC and non-O157 EHEC strains express lymphostatin (also known as LifA), a chromosomally encoded 365-kDa protein. We previously demonstrated that lymphostatin is a putative glycosyltransferase that is important in intestinal colonization of cattle by EHEC serogroup O5, O111, and O26 strains. However, the nature and consequences of the interaction between lymphostatin and immune cells from the bovine host are ill defined. Using purified recombinant protein, we demonstrated that lymphostatin inhibits mitogen-activated proliferation of bovine T cells and, to a lesser extent, proliferation of cytokine-stimulated B cells, but not NK cells. It broadly affected the T cell compartment, inhibiting all cell subsets (CD4, CD8, WC-1, and γδ T cell receptor [γδ-TCR]) and cytokines examined (interleukin 2 [IL-2], IL-4, IL-10, IL-17A, and gamma interferon [IFN-γ]) and rendered T cells refractory to mitogen for a least 18 h after transient exposure. Lymphostatin was also able to inhibit proliferation of T cells stimulated by IL-2 and by antigen presentation using a Theileria-transformed cell line and autologous T cells from Theileria-infected cattle. We conclude that lymphostatin is likely to act early in T cell activation, as stimulation of T cells with concanavalin A, but not phorbol 12-myristate 13-acetate combined with ionomycin, was inhibited. Finally, a homologue of lymphostatin from E. coli O157:H7 (ToxB; L7095) was also found to possess comparable inhibitory activity against T cells, indicating a potentially conserved strategy for interference in adaptive responses by attaching and effacing E. coli.