Performing Department
Entomology
Non Technical Summary
Bovine anaplasmosis is considered the most critical disease in cattle worldwide. It is caused by the intracellular bacterium,Anaplasma marginale, that infects erythrocytes, leading to anemia, weight loss, decrease in milk production, spontaneous abortions, and death.A. marginaleinfections can result in mortality of naïve cattle at rates of up to 30%. The presence or introduction of persistently infected animals into a production system with naïve animals can have significant negative economic consequences in herds where ticks are present. This disease represents a threat to the cattle industry in the US, which brings ~$77 billion in sales. It is also a risk for food security as milk and meat are important sources of protein. The estimated losses in the US are over $300 million per year, and the cost of treating bovine anaplasmosis is projected to be over $400 per animal. In the US,A. marginaleis commonly found in Southern, Midwestern, and Western states but has been reported in every state. The exact prevalence of the disease in the US is unknown; however, Georgia, Kentucky, and Texas have reported estimates of true seroprevalences of 2.62%, 10.3%, and 15.91%, respectively. One characteristic of the disease is that animals become permanent carriers after they recover from acute infection. Thus, finding alternative mechanisms to prevent and treat bovine anaplasmosis is critical.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Bovine Anaplasmosis is a tick-borne disease that results in estimated losses of over 300 million USD per year in the US. Bovine Anaplasmosis is caused by the obligate intracellular pathogen Anaplasma marginale, which infects red blood cells, resulting in anemia, fever, weight loss, decreased milk production, spontaneous abortions, and, in naïve herds, can cause up to 30% mortality. Treatment cost can be over 400 USD per animal in the US, imposing a financial burden to cattle producers. Recent studies indicate that current treatments may not be effective and no commercial vaccine is available in the US. The development of an effective vaccine is needed, but it is hindered by several factors: 1) the ability of pathogen to escape cattle's immune responses, 2) the capacity of tick saliva to hamper the cow's immune response and 3) the ability of the bacteria to enhance the detrimental effects of tick's saliva on the immune system of the animal.The aim of theseprojects is to understand how A. marginale affects bovine skin immune responses and alters the immune gene expression at the bite site.The long-term goal of our project is to define the factors that are important for the transmission and establishment of bovine. We will identify the host immune components that lead to protection. With this knowledge, it is likely that applied scientist will be able to develop vaccines or treatments to this disease.The shor-term goalsof this project areto: 1) determine the changes in immune response at the tick feeding site during pathogen transmissionand 2) characterize the changes in salivary secretions during the transmission ofA. marginale.Through the development of a more complete understanding of the tick-pathogen-host interactions at the time of initial infection, we will identify additional antigens and mechanisms that could be leveraged for the development of a vaccine that protects against A. marginale infection in cattle.
Project Methods
Aim 1: Determine the changes in immune response at the tick feeding site during pathogen transmissionChanges in immune gene expression at during transmission of A. marginale: Calves over 45 kg will be injected with blood from splenectomized animal with over 1x109 organism/ml obtained from collaborators at Washington State University. Infection status will be monitored by light microscopy of blood smears stained using Giemsa's stain. Blood samples will be taken from the jugular every other day vein and Percentage of Infected Erythrocytes (PPE) and the Packed Cell Volume (PVC) will be measure. When blood smears show a PPE of 0.5%, samples will be sent to TVDML to confirm infections. Animals will be monitored until they reach PPE of 10%. Male Dermacentor andersoni ticks will be placed on animals for pathogen acquisition and allow to feed for 7 days. Ticks will be recovered from the animals and placed at 90% RH 16:8 L:D cycle for 7 days to avoid mechanical transmission. Infection status of the ticks will be confirmed using primers against the single gene encoding the Major Surface Protein 5 (Msp5). Ticks will be used to infest 4 naïve animals. Infected and uninfected ticks will be placed in the opposite side of the animals. This will allow us to compare the changes in gene expression that occur during transmission versus tick feeding, while eliminating changes due to the genetic differences between animals. Fifty infected and uninfected males will be placed on naïve animals within pre-made ethylene-vinyl acetate (EVA) foam capsules glued to the side of the animals. This will prevent the ticks from feeding in cluster due to the secretion of aggregation pheromones. Ticks will be allowed to feed for 7 days and five skin samples will be taken from the site of tick feeding while the animals are under deep anesthesia to avoid cell apoptosis due to lack of oxygen. Skin samples also be taken from sites without ticks to determine the baseline level of gene expression in each animal, resulting in a total of 20 skin samples per animal. This will allow us to eliminate samples with low RIN value. Skin samples will be placed in RNAlater and RNA will be isolated after removing the tick for RNA-seq. Eukaryotic mRNA will enriched by poly-A tail selection. Samples will be sent to GENEWIZ for sequencing. Differential Gene Expression (DGE) between skin samples within and between animals will be determined. Those genes that are consistently differentially expressed in all animals will be used to define whether specific immune pathways are enriched during A. marginale transmission using Reactome.Analysis of the changes in cytokine, chemokine, and growth factors at the site of transmission: Eight (8) skin samples from sites feeding upon by infected and uninfected ticks will be used to determine alterations in cytokine, chemokine, and growth factors using Bovine Cytokine Arrays commercially available through Raybiotech. Skin samples from sites without ticks will be used to determine the baseline levels of cytokine in the skin. Skin samples will be placed in dry ice after harvesting and will be immediately homogenized in the laboratory using protocols already established (13). We will focus in chemokines and cytokines involved in recruitment of neutrophils, basophils, mast cells, and T cells, including γδ T-cells. We will also look at growth factors since vesicles appear to act on dealing wound healing responses (13, 17). Differences in cytokine, chemokine, and growth factor levels in the skin will be evaluated by One-way ANOVA.Determine changes in immune cell at the skin duringA. marginaletransmission: Animals and ticks will be infected as described above. Fifty (50) infected and uninfected ticks will be infested in three naïve animals in groups of three ticks per capsule and ticks will be allowed to feed for 7 days as describe above. Skin samples (~10 from each treatment and each animal) will be taken and place in RPMI on ice. Single cell suspension will be prepared using the protocol that we developed in collaboration with Dr. Davis at Washington State University. Briefly, skin will be dissociated with the Whole Skin Dissociation kit (Miltenyibiotec) and pass through a 70 μm filter. Cells will be separated from cell debris using Ficoll-Percol. Cells will be labeled using monoclonal antibodies against WC1, CD3, γδ T-cells, CD45RO, and Neutrophils. Cells will be quantified using FACS and the differences in population frequencies will be evaluated by One-way ANOVA. The remaining skin samples will be used to validated the FACS results using ImmunoHistoChemistry (IHC) and HE staining. Five (5) skin samples from infected and uninfected tick feeding sites will placed in 10% formalin for parafilm embedding and HE staining at the Histology lab at TAMU, while other 5 skin samples will place in SakuraOCT 4583cryo embeddingcompound and flash freeze it in liquid nitrogen. Samples will be cut to 5 μm sections and labeled at the Immunochemistry lab (IHC) using the same antibodies described above and compared to commercially available antibodies. Cells will be visualized by fluorescent microscopy at the Microscopy and Imaging Center (MIC). Pictures will be analyzed using ImageJ to determine differences in Arbitrary Fluorescence Units (AFU) between treatments (13). Differences will be evaluated by One-way ANOVA.Aim 2: Characterize the changes in salivary secretions during the transmission ofA. marginaleDefine the alterations in extracellular vesicle biogenesis in tick by tick-borne intracellular bacteria: The salivary glands from infected and uninfected ticks recovered from Aim 1 will be dissected and pull in groups of 10. RNA will be isolated and will be send to the University of Minnesota. We will reconstruct the whole biogenesis pathway in D. andersoni as previously described (13). These sequences will be used to selectively sequence and quantify the RNA associated with these genes. Nanopore selective sequencing allows to reject specific molecules as they are being sequencing, thus allowing to increase coverage in the sequences of interest (18). The differences in expression of genes involved in the secretion of exosomes and microvesicles will be evaluated to identify specific targets that are affected by the bacterial infection.Characterize the changes in the proteomic cargo of extracellular vesicles duringA. marginaletransmission: Infected and uninfected ticks will be placed on naïve animals as described above. Ticks will be allowed to feed for 7 days and removed. Salivary glands will be dissected out and placed in extracellular vesicles-free tick media as previously described (13). Vesicles will be isolated by a series of differential centrifugations followed by overnight ultracentrifugation. Vesicles will be separated from soluble proteins by size exclusion using 300 kDa MW microfuge filters. Vesicles will be resuspended in PBS and submitted for Parallel Accumulation Serial Fragmentation (PASEF) MS/MS spectrometry using a timsTOF pro at the Charles W. Gehrke Proteomics Center at the University of Missouri. We have already worked with Dr. Mooney at University of Missouri in other projects. The quantitative differences in proteins between treatments will be assessed, using label-free quantification (LFQ) in PEAKS. The proteins that show quantitative differences will be used to determine whether specific pathways are particularly enriched within these vesicles.