Exploratory Investigations in Immunology & Infectious Diseases

<p>NON-TECHNICAL SUMMARY:<br/> Infectious disease causes considerable loss for livestock producers by reducing production of animal units and by reduced sales because of food safety concerns. Immunology and Infectious Diseases (IMID) is the only research unit in Montana focused on animal health, particularly on the study of infectious diseases of cattle. New faculty members joining IMID are required to initiate new research projects. In addition, other faculty not on Montana Agricultural Experiment Station (MAES) funding may be hired to develop new short-term projects. These projects are in support of the respective missions of MAES and IMID. This departmental project is to be used by these scientists prior to their approved MAES project. Support is also provided to maintain and operate departmental research facilities.
<p>APPROACH:<br/> IMID scientists utilize state-of-the-art molecular approaches to address basic and applied problems in infectious disease research. These research programs require laboratories, large and small animal facilities, clinics, and modern research equipment, such as flow cytometers, DNA sequencers, and genomics analysis facilities. Specific methods and procedures utilized are dependent upon program type and necessary protocols. New or existing faculty members must develop an understanding of Montana and regional issues and a more in-depth understanding of existing research programs, prior to developing their own MAES project proposals.
<p>PROGRESS:<br/> 2012/01 TO 2012/12OUTPUTS: Funds were used to support seed projects for two current faculty members and start-up of a new faculty member in the department. Dr. Josh Obar is investigating the role of mast cells in the pathogenesis of listeria infection. Listeria monocytogenes causes widespread human and animal disease. In humans, Listeria is one of the most common causes of food borne illnesses. In ruminants, it can cause encephalitis, septicemia, late-term abortions, and on rare occasions mastitis. This project is focused on investigating the hypothesis that mast cells are critical mediators in the detrimental outcome of Listeria infection. Dr. Benfang Lei is investigating virulence factors and protective antigens among uncharacterized cell wall-linked proteins of Streptococcus equi, the pathogen that causes strangles. His current work is focused on
generating S. equi mutants. Ultimately, these studies could lead to target antigens for vaccine development to treat strangles. Dr. Blake Wiedenheft is a new faculty member in the department. His research focuses on investigating adaptive immune systems in bacteria. Bacteria and archaea acquire resistance to viral and plasmid challengers by integrating short fragments of foreign nucleic acids into the host chromosome at one end of a repetitive element known as a CRISPR (clustered regularly interspaced short palindromic repeat). This research is focused on understanding the mechanisms of CRISPR RNA-guided recognition and destruction of invading nucleic acids by the immune system in Pseudomonas aeruginosa (PA14). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS:
Nothing significant to report during this reporting period.
<p>PROGRESS: 2011/01/01 TO 2011/12/31<br/>OUTPUTS: Funds were used to support a seed project for one junior faculty member in the department. Dr. Josh Obar is a new faculty member in the department and began a project investigating the role of mast cells in the pathogenesis of listeria infection. Listeria monocytogenes causes widespread human and animal disease. In humans, Listeria is one of the most common causes of food borne illnesses. In ruminants, it can cause encephalitis, septicemia, late-term abortions, and on rare occasions mastitis. This project is focused on investigating the hypothesis that mast cells are critical mediators in the detrimental outcome of Listeria infection. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing
significant to report during this reporting period.
<p>PROGRESS: 2010/01/01 TO 2010/12/31<br/>OUTPUTS: Funds were used to support seed projects for three junior faculty members in the department. Project 1: Dr. Jovanka Voyich-Kane investigated the expression of bovine chemokines that regulate immune cell homing to tissues. Chemokines are chemotactic for cells expressing the appropriate chemokine receptors, and the tissue-specific expression of chemokines is essential to the generation of adaptive immune responses. The chemokines CCL25 and CCL28 are of particular interest to mucosal immunology, and CCL28 has been shown to exhibit potent antimicrobial activity against various bacteria. Although chemokines and chemokine receptors have been shown to play key roles in immune function, the tissue distribution of CCL25 and CCL28 in the bovine system remains uncharacterized. Given the potential importance of these chemokines in
mucosal immunity, this project focused on characterization of the expression patterns of bovine CCL25 and CCL28 and their principal ligands CCR9 and CCR10. Project 2: Dr. Josh Obar is a new faculty member in the department and began a project investigating the role of mast cells in the pathogenesis of listeria infection. Listeria monocytogenes causes widespread human and animal disease. In humans, listeria is one of the most common causes of food borne illnesses. In ruminants, it can cause encephalitis, septicemia, late-term abortions, and on rare occasions mastitis in ruminants. This project is focused on investigating the hypothesis that mast cells are critical mediators in the detrimental outcome of listeria infection. Project 3: Dr. Robert Cramer investigated the role of Nosema infection in death of honey bee colonies. Specifically, their research is focused on understanding the
mechanisims used by the microsporidian N. ceranae to infect honeybees and the association of Nosema infection with DNA virus infection of honeybees. Their current research now focuses on understanding how proteins present on the surface of the Nosema polar tube directly interact with proteins on the surface of the honeybee gut epithelial cells to facilitate infection. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
<p>PROGRESS: 2009/01/01 TO 2009/12/31<br/>OUTPUTS: Funds were used to support seed projects for three junior faculty members in VMB. Dr. Jovanka Voyich-Kane investigated Staphylococcus aureus isolates in horses. Methicillin-resistant Staphylococcus aureus (MRSA) is an important bacterium in human medicine that is becoming increasingly more significant in veterinary medicine. In recent years, reports of infection and colonization in horses have increased. This study was conducted to identify S. aureus among horses and horse personnel residing on guest ranches. Seven guest ranches in southwest Montana were chosen. This study identified healthy horses colonized with S. aureus, including one MRSA strain, E046. Initial characterization of equine E046 has demonstrated that it contains SCCmec type II, typical of hospital-associated S. aureus. Dr. Jay Radke studied gene
expression in bovine monocytes infected with Mycobacterium avium ssp. paratuberculosis. Mycobacterium avium ssp. paratuberculosis infection in cattle causes the inflammatory bowel condition known as Johnes disease. The host response to infection is typified by uptake of bacteria in macrophages and the subsequent induction of antigen-dependent T cell-mediated secretion of pro-inflammatory cytokines, such as intereferon gamma (IFN). This group characterized differences in the bovine lymphocyte response to in vitro infection and report consistent, robust proliferation of naive gamma/delta and CD8+ T cells after co-culture with macrophages that had taken up avirulent M. avium ssp. avium, but not the virulent M. avium ssp. paratuberculosis. T cell subset depletions and two-color flow cytometry analyses verified that IFN production came predominately from proliferating CD8+ T cells. This
induced IFN was further correlated with increased killing of M. avium ssp. avium by infected macrophages. Robert Cramer investigated the role of aspergillus infection in death of honey bee colonies. The major completed goal of this project has been the establishment of honeybee research infrastructure at MSU. We have established a small apiary near the campus. This facility fostered the development of in vitro cage trials with live honeybees and the emerging fungal-like pathogen Nosema ceranae. Specifically, we continue to explore the hypothesis that a synergistic interaction between N. ceranae and an unknown large DNA virus are contributing to bee losses across the country. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this
reporting period.
<p>PROGRESS: 2008/01/01 TO 2008/12/31<br/>OUTPUTS: Funds were used to support seed projects for three junior faculty members in VMB. Dr. Jovanka Voyich-Kane investigated the strain-specific identification of Staphylococcus aureus isolates causing mastitis in Montana Dairy farms. Dr. Jay Radke studied gene expression in bovine monocytes infected with Mycobacterium avium ssp. paratuberculosis. Dr. Robert Cramer investigated the role of aspergillus infection in death of honey bee colonies. Dr. Cramer's lab proposed to developed a polymerase chain reaction diagnostic tool for identifying Nosema infections in honeybees that can differentiate between N. ceranae and N. apis. This screening tool was used to assess the presence of N. ceranae in honeybee colonies around Montana and the Pacific Northwest. PARTICIPANTS: Nothing significant to report during this reporting period.
TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
<p>PROGRESS: 2007/01/01 TO 2007/12/31<br/>Funds were used to support seed projects for three junior faculty members in VMB. Dr. Jovanka Voyich is pursuing the strain specific identification of Staphylococcus aureus isolates causing mastitis in Montana Dairy Farms. Dr. Jay Radke is studying gene expression in bovine monocytes infected with Mycobacterium avium ssp. paratuberculosis. Dr. Robert Cramer is investigating the role of aspergillus infection in death of honey bee colonies.
<p>PROGRESS: 2006/01/01 TO 2006/12/31<br/>Funds were used to support seed projects for four junior faculty members in VMB. Dr. Jovanka Voyich is pursuing the strain specific identification of Staphylococcus aureus isolates causing mastitis in Montana Dairy Farms. Dr. Jay Radke is studying gene expression in bovine monocytes infected with Mycobacterium avium ssp. paratuberculosis. Dr. Ben Lei is characterizing proteins in Streptococcus equi. Dr. William Halford is investigating interferon-sensitive herpes viruses. Funds were also used to repair the roof and cabinets in the large animal clinic.
<p>PROGRESS: 2005/01/01 TO 2005/12/31<br/>Funds were used for two new VMB faculty members. Dr. Ben Lei advanced a project in determining antigenic targets for Streptoccocus equi, the causative agent of horse strangles. To date, 6 antigens have been identified and mutant bacteria produced. Studies utilizing these mutants in a mouse model of strangles are in progress. Dr. William Halford is developing a research project to determine determine if host interferons are pivotal in the regulation of viral latency of animal alphaherpesviruses such as bovine herpesvirus 1 (BHV-1) and Marek's disease Virus (MDV).
<p>PROGRESS: 2004/01/01 TO 2004/12/31<br/>Funds were used for three new VMB faculty members. Dr. Ben Lei began a project in determining antigenic targets for Streptoccocus equi, the causative agent of horse strangles. To date, 6 antigens have been identified and mutant bacteria produced. Studies utilizing these mutants in a mouse model of strangles are in progress. Dr. Richard Bessen has now established his lab at VMB and has initiated experiments that will determine the mechanisms by which prions (such as those that cause BSE or CWD)translocate to the central nervous system after they are ingested. Dr. William Halford just recently arrived at VMB and has just begun to set up his lab. He is in the process of developing a research project that will investigate the mechanisms involved in chronic bovine herpes virus infections.
<p>PROGRESS: 2003/01/01 TO 2003/12/31<br/>Funds were used to establish the laboratory of a new faculty member of this Department, Benfang Lei. Dr. Lei has made excellent progress in beginning to investigate Streptococcus equi, the causative agent of horse strangles. Dr. Lei has cloned six proteins of S. equi and is purifying those proteins. Next year he will test the efficacy of these proteins as vaccine candidates utilizing a mouse model of S. equi infection. Funds were also used to establish the laboratory of a second new faculty member of this Department, Richard Bessen. Dr. Bessen arrived just last October and has just begun his research efforts. His project is in Chronic Wasting disease and he will concentrate on studies of the pathogenesis and transmission of this disease.
<p>PROGRESS: 2002/01/01 TO 2002/12/31<br/>My laboratory has much experience in the investigation of respiratory immunology of mouse models of human disease. We have begun to adapt the technologies used in these past studies to better understand respiratory immunology in calves. We have developed the technique of fiberoptic brochoscopy in calves so that now we can inoculate specific airways of calves with BVD virus. We can then go back to the same lung airway in an individual calf multiple times after inoculation and either biopsy the airway through the bronchoscope or lavage the airway with sterile saline. We can then aspirate back the saline and analyze the recovered fluid for immune cells that have accumulated in the airways in response to the viral infection. We can also analyze the fluid for viable virus and in this way follow the proliferation and clearance of the
virus together with the host response. Cells in the recovered lavage fluids are then analyzed by flow cytometry to determine the phenotype and activation state of the responding immune cells. The fluid can also be analyzed for levels of different isotypes of antibodies. Lastly, we have developed a technique to biopsy the tonsils of infected calves. We can then analyze the immune cells recovered from the tonsils in order to study the role of this tissue in the immune response to the virus. Thus, we have developed powerful techniques to investigate the kinetics of immune responses to viruses in the respiratory tract of calves. We are now poised to utilize these techniques to better understand bovine immunity to respiratory tract viruses.
<p>PROGRESS: 2001/01/01 TO 2001/12/31<br/>(Dr. A. Harmsen)Our efforts toward establishing a research program to study the pathogenesis of and immunity to bovine respiratory disease are underway. We have successfully developed the techniques for obtaining bronchoalveolar lavage cells from cattle lungs for the study of lymphocyte subpopulations and cytokine profiles. Protocols are being developed to determine lymphocyte cell traffic into the lung airways in response to the bacterial and viral agents relevant to bovine respiratory disease. (Dr. Ed Schmidt)Targeted mutagenesis can be used to create genetically modified animals; however, to date, other than one reported success in sheep, there are only published reports of success in mice. The proposed research is aimed at increasing the efficiency of targeted mutagenesis in bovine cells, which could in turn be used to create
animals by cloning. We propose to produce bovine cell lines from a pure-bred breed of dairy cattle (American Holstein) and from an unrelated breed (free-range beef cattle from Montana). DNA from the Holstein cells will be used to produce vectors for targeted mutagenesis. Differences in targeting efficiency between the cell lines will be correlated to the number of DNA sequence differences between the two cell lines at the targeting site. We will then introduce mutations into the targeting vector and measure the effects of these on targeting efficiency in the two cell lines. As the first systematic study to analyze parameters affecting targeted mutagenesis in bovine cells, we have produced a line of bovine embryonic fibroblast cell (BEF) from a single 40 day-old bovine fetus that we harvested from a 'pure-bred' American Holstein heifer inseminated with pure American Holstein semen. The
rationale for this breed choice was to minimize heterozygosity in the resultant fetus. From the same fetus, we prepared a genomic DNA library in lambda phage. We expanded the BEF cell line to second passage, and froze several hundred vials of these early-passage cells in liquid nitrogen to provide an essentially permanent stock of BEFs that genetically match our genomic library perfectly. Characterization of the library indicate the primary library had >10-fold coverage of the genome with an average insert size of over 22kb. We have screened this library and isolated 5 pure clones of the bovine tbp gene, which will form the target for our studies. We chose this gene as a representative paradigm largely because we have great experience in working with targeting this gene in mouse cells. This is the first year of this study and it is still in the development stage. Ongoing work is aimed at
producing genetically disparate BEF lines and in creating defined targeting vectors from our clones. Future development work will involve establishing transfection conditions, genetic analyses, and cell cloning technology that will work in this system. At that point, we can begin actually measuring the parameters that affect gene targeting in bovine cells.
<p>PROGRESS: 2000/01/01 TO 2000/12/31<br/>A new program focused on understanding mechanisms of gene regulation was initiated. This is a new project is focused on gene targeting in cattle. Dr. Edward Schmidt, an expert in gene targeting technology, has joined our faculty and will be the primary investigator on this program. Current work is focused on producing a bovine embryo fibroblast cell line that can be used for gene targeting. Several cell lines have been produced and are now being characterized genetically.
<p>PROGRESS: 1999/01/01 TO 1999/12/31<br/>The overall goal of Dr. Speer's project is identify the parasite surface and internal molecules involved in the penetration and development of Sarcocystis neurona into host cells. Monoclonal antibody reagents are being used 1) to characterize S. neurona antigens by immunoprecipitation and Western blot analysis; 2) determine the functional roles of parasite antigens in parasite penetration and development; and 3) determine the ultrastructural localization of parasite antigens during parasite development. To date, we have found some monoclonal antibodies that are capable of inhibiting parasite development and multiplication. The goal of Dr. Hardy's project is to understand the role of rotavirus nonstructural protein NSP1 in virus replication and cytopathology. The specific aims of this project were 1) to clone the genes encoding
NSP1 for two bovine rotavirus strains, 2) to produce immunologic reagents to NSP1, and 3) to establish cell lines inducible for expression of NSP1. We have completed cloning and sequencing of NSP1 of bovine rotavirus strain B641 and expressed recombinant NSP1 in bacteria. Purified NSP1 then was used to immunize mice for polyclonal antibody production. We have obtained a high titer serum that reacts specifically with NSP1 in rotavirus-infected cells by Western blot. We also have isolated stably transfected MA104 cell lines inducible for expression of a luciferase reporter gene. Isolation of cell lines inducible for NSP1 is in progress. Ongoing studies include co-immunoprecipitation and cross-linking analyses of NSP1 in infected cells to identify host cell proteins that interact with NSP1. We also have recently obtained data to suggest that NSP1 is involved in regulating rotavirus gene
expression at the level of protein synthesis. These data are important in determining how the viral genome is efficiently replicated. By understanding these processes at the molecular level, new therapeutic strategies to inhibit bovine rotavirus replication in the host can be developed. Dr. Schmidt's project aims to establish methodology for purifying homogeneous populations of spermatids representing different stages along the maturation pathway. Mammalian spermatozoa are highly specialized for performing as efficient fertilization vectors. To allow efficient swimming and motility, nucleus of spermatid is streamlined by densely compacting the chromatin. This compaction is incompatible with transcriptional gene expression. Rather, spermatid maturation occurs in the absence of transcription by translating mRNAs that were synthesized earlier and stored as mRNP particles. This technology
will be valuable for future investigations of the molecular mechanisms of translational regulation during spermiogenesis. Our approach is to use a fluorescence-activated cell sorting (FACS)-based method of purifying sub-populations of spermatids from transgenic mice that we have made which express GFP exclusively in their spermatids. We have established methods to explant spermatogenic cells from the testes of these GFP-expressing mice that maintain the integrity of cellular mRNA.
<p>PROGRESS: 1998/01/01 TO 1998/12/31<br/>This project supported the research efforts of several faculty members in Veterinary Molecular Biology. Dr. Michele Hardy has established a nationally competitive research program on livestock bovine enteric viruses. Dr. Hardy has submitted grants to the USDA, NSF, and NIH for additional support. Funds from this project also supported general departmental efforts in studies of bovine immunology (Jutila) and protozoan infection of livestock (Speer). Speer tested several cell lines for their ability to support the development of schizonts and merozoites of two isolates of Sarcocystis neurona and Sarcocystis falcatula. The greatest numbers of merozoites were produced in bovine monocytes (M617 cells), moderate numbers in VERO cells and low numbers in bovine pulmonary artery endothelial cells (CPA). Schizogony occurred in rat myoblasts
(L6) infected with S. neurona, but not S. falcatula. Merozoites of S. falcatula developed in quail myoblasts (QM7), but not in rat myoblasts. Neither parasite developed to sarcocysts in any of the cell types tested. The development of schizonts and merozoites of S. neurona and S. falcatula was also studied ultrastructurally. We also discovered a third species of Sarcocystis in opossums which we studied in immunodeficient mice and by transmission electron microscopy.
<p>PROGRESS: 1997/01/01 TO 1997/12/31<br/>This project supported the research efforts of two new faculty members in Veterinary Molecular Biology (Drs. Michele Hardy and David Pascual). Dr. Pascual has established a nationally competitive research program on livestock vaccines. He has demonstrated the effectiveness of Salmonella vectors in delivering E. coli vaccine antigens to the gut mucosa. He has received funding from the USDA NRI for this work. Dr. Hardy started her position last summer and Hatch support has enabled her to establish a new research program on bovine enteric viruses. Dr. Hardy has submitted grants to the USDA, NSF, and NIH for additional support. Funds from this project also supported general departmental efforts in studies of bovine immunology and protozoan infection of livestock.
<p>PROGRESS: 1996/01 TO 1996/12<br/>This project supported the reseach efforts of 3 different faculty in Veterinary Molecular Biology in the past year. Dr. C.A. Speer's research on Toxoplasma expanded on his characterization of a unique pathway of development in host cells. Dr. Speer started a separate Hatch project this past Fall. Dr. Mark Quinn's research is on the NADPH oxidase system of bovine and human neutrophils. He has also established a separate Hatch project for his studies. Dr. David Pascual is a new faculty member. In the past year he has established a new vaccine development program for cattle. He acquired an NRI competitive grant to help initiate his studies and will begin a new Hatch project next year. This project has also funded general departmental projects related to animal care and experimentation. Livestock diseases being studies include
trichomoniasis, coccidiosis, toxoplasmosis, and e. coli-induced enteritis.
<p>PROGRESS: 1995/01 TO 1995/12<br/>This project supported the research efforts of 3 different faculty in VeterinaryMolecular Biology in the past year. Dr. C.A. Speer's research on Toxoplasma led to the identification of a unique pathway of development in host cells. Dr. M.T. Quinn's research is on the NADPH oxidase system of bovine and human neutrophils. He has recently been awarded an NRI-USDA grant for his work and has established a separate Hatch Project for his studies. Dr. D. Pascual started in our department this past Fall. Funds from this project have aided his research on vaccine delivery systems for cattle. This project has also provided support for general departmental projects related to animal care and experimentation.
<p>PROGRESS: 1994/01 TO 1994/12<br/>Several cell types were examined for their ability to support in vitro development of the tachyzoites of the NC-1 isolate of Neospora caninum. Bovine cardiopulmonary artery endothelial (CPA), embryonic bovine trachea (EBTr), Madin-Darby bovine kidney (MDBK), and whole mouse embryo (3T3) cells were grown in a serum-free medium (HyQ-CCMI, Hyclone, Logan, UT), inoculated with tachyzoites and monitored for parasite development at daily intervals for as long as 18 days. Although all four cell types supported the development of substantial numbers of parasites, the best development occurred in CPA cells in which 50-200 million tachyzoites were produced daily from each of several 75 sq cm flask at 8 to 11 days after inoculation. In vitro-produced tachyzoites are being tested for efficacy in a diagnostic assay. Genomic and cDNA libraries are
being generated from in vitro-produced tachyzoites.
<p>PROGRESS: 1993/01 TO 1993/12<br/>The seasonal variations of an outbreak of Sphaeridiotrema globulus in migratory waterfowl has been studied. The outbreak occurred on two small lakes near Billings, MT. Regular collections of the snail vector, Bythinia tentaculata, have been made and the parasite populations have been characterized. The study results indicate a dense parasite population with potential for future outbreaks, especially during waterfowl migration seasons. The parasite populations in a free-ranging bison herd have been characterized by regular fecal examinations. Several helminth and protozoan parasite species are found throughout the year, including: Nematodirus spp., Eimeria spp., Trichuris spp. several unidentified gastrointestinal nematodes, and Dictyocaulus spp. No clinical manifestations of parasitisms have been observed.
<p>PROGRESS: 1992/01 TO 1992/12<br/>Evaluated blood analysis instrument (Quantitative Buffy Coat Analysis, QBCA) forovine complete blood cell counts. Results should allow inexpensive analysis of ovine blood (in-house) for the average livestock veterinarian. Evaluated chemical castration in the boar, preliminary stages only, current compounds are unacceptable. Continued work on biofilm growth on metallic implants (chrome cobalt and titanium alloys) - Pseudomonas culture evaluation of antiarrythmic pharmaceuticals on atrial and ventricular arrythemias in the horse (naturally occurring cases).
<p>PROGRESS: 1990/10 TO 1991/09<br/>1) A cDNA library was derived from sporulated oocysts of Cryptosporidium parvum.Numerous monoclonal antibodies have been generated against sporulated oocysts and sporozoites of C. parvum, and some of these have been partially characterized. 2) The distribution and seasonal transmission patterns for bovine fascioliasis have been established for nearly all of Montana. 3) A highly specific and sensitive oligonucleotide probe has been developed to detect Fasciola hepatica miracidia in snails. 4) A highly specific and sensitive DNA probe was developed for the diagnosis of bovine trichomoniasis. 5) Various species of Sarcocystis were studied in horses and dogs.
<p>PROGRESS: 1990/01 TO 1990/12<br/>1) Efforts continued to produce DNA-probes for detecting fasciola larvae in snail vectors. Studies were initiated to generate monoclonal antibodies against various stages of Fasciola that occur in the mammalian host. 2) Studies were initiated to characterize the surface antigens on Candida albicans and on various types of endothelial cells of sheep, cattle and goats. These studies consisted of functional assays and monoclonal antibody production. 3) Efforts to obtain total RNA and to develop a cDNA library of Cryptosporidium parvum were also started during the year. 4) Fatal cutaneous and visceral infections due to a Sarcocystis-like organism was characterized in Rottweiler dogs.
<p>PROGRESS: 1989/01 TO 1989/12--Efforts to produce a DNA-probe for detecting Fasciola larvae in the snail vector have progressed to the sequencing stage. Isoelectric focusing (IEF) experiments as a means of early detection of Fasciola infection in the snail an as a means of snail species differentiation are also being done. The initial experiments revealed a definite difference in the protein banding patterns between infected and non-infected snails. --Sporozoites, merozoites and bradyzoites of Sarcocystis cruzi were found to share several polypeptides and antigens with similar molecular weights but each also had unique polypeptides. Merozoites harvested from cultured cells at 29 to 60 days after inoculation of sporozoites exhibited temporal variation in expression of certain polypeptides --The ultrastructural characteristics were described for sporozoites and
zoites of Hammondia heydorni and for tachyzoites, bradyzoites and tissue cysts of Neospora caninum. --Monoclonal antibodies and immunoelectron microscopy were used to study the ultrastructural localization antigenic sites on various stages of Eimeria acervulina and Eimeria tenella.
<p>PROGRESS: 1989/01 TO 1989/12<br/>--Efforts to produce a DNA-probe for detecting Fasciola larvae in the snail vector have progressed to the sequencing stage. Isoelectric focusing (IEF) experiments as a means of early detection of Fasciola infection in the snail an as a means of snail species differentiation are also being done. The initial experiments revealed a definite difference in the protein banding patterns between infected and non-infected snails. --Sporozoites, merozoites and bradyzoites of Sarcocystis cruzi were found to share several polypeptides and antigens with similar molecular weights but each also had unique polypeptides. Merozoites harvested from cultured cells at 29 to 60 days after inoculation of sporozoites exhibited temporal variation in expression of certain polypeptides --The ultrastructural characteristics were described for sporozoites and
zoites of Hammondia heydorni and for tachyzoites, bradyzoites and tissue cysts of Neospora caninum.
<p>PROGRESS: 1988/01 TO 1988/12<br/>
A newly identified coccidian parasite (Neospora caninum) was found to cause fatal neurological disease in dogs. N. caninum was ultrastructurally distinct from other cyst-forming coccidia. An in vitro system based on long-term cultures of bovine pulmonary artery endothelial cells was developed to culture the vascular phase (merozoites) of several Sarcocystis species of livestock. This in vitro system plus antigen analysis of merozoites helped deduce a possible mechanism for the vascular damage (e.g., petechial hemorrhages, vasculitis and perivascular mononuclear infiltration) commonly observed in Sarcocystis infections. An in vitro system was developed for the cultivation of Hammondia heydorni, a coccidian parasite of dogs. A survey revealed that more than 90% of the sheep in the United States are infected with Sarcocystis. The sarcocyst walls of four different Sarcocystis species (i.e., S. arieticanis, S. gigantea, S. medusiformis and S. tenella) in sheep were found to be ultrastructurally distinct. A survey of fascioliasis revealed that 50% of the calves from Victor, Montana were infected. A Dot-ELISA test on the sera from these calves gave only a 50% correlation with fluke-egg detection via fecal examination.
<p>PROGRESS: 1987/01 TO 1987/12<br/>
An end point dilution microtitration assay was developed that can be used for titration of both cytopathic and noncytopathic isolates of bovine virus diarrhea-mucosal disease virus (BVDV). IFA was used for the detection of BVDV-infected MDBK cells. The virus titer was derived using the Poisson distribution from the number of uninfected wells of Terasaki plates. Antigens of Eimeria bovis were studied for their ability to cause lymphocyte transformation in vitro. An antigen, called P20, with a molecular weight of 20,000, was detected in western blots with polyclonal immune serum and with two monoclonal antibodies. Treatment of E. bovis sporozoites with polyclonal or certain monoclonal antibodies caused nearly an 80% reduction in penetration of MDBK cells in vitro. Antigens of Sarcocystis cruzi merozoites, bradyzoites and sporozoites were studied by SDS-PAGE, iodination, western blots, and monoclonal antibodies.
<p>PROGRESS: 1986/01 TO 1986/12<br/>
Significant findings on Bacteroides fragilis include (l) a higher percentage of bacteriocinogeny among the enterotoxigenic isolates as compared to the nonenterotoxigenic isolates, (2) a low percentage of lysogeny among all isolates, (3) the isolation of temperate phages from lysogenic strains and from fecal specimens which are able to lysogenize a limited number of phage hosts and (4) preliminary data showing that the supernatants of enterotoxigenic B. fragilis are cytotoxic for some cell lines. A pilot project was designed to determine the role of mast cells (MC) in fracture healing, a major complication of osteopetrosis (OP). The following four mutant strains of rats and mice were used: nude mice, W/W(v) mice, toothless (TL) rats and osteopetrotic (OP) rats. Experimental, closed, transverse fractures of the left distal femoral metaphysis were induced in anesthetized rats and mice. Randomly selected animals were euthanized at 30, 60 and 90 days. TL and OP rats had large calluses, delayed malunion fracture sites and abundant cartilage production. Nude and W/W(v) mice had radiographic and histologic healing phases indistinguishable from controls. These preliminary results suggest that: (l) MC are not essential to normal fracture healing; (2) excessive numbers of MC may retard fracture healing; and (3) absence of a thymux has no discernable effect on fracture healing.
<p>PROGRESS: 1984/10 TO 1985/09<br/>
This project is used for initial or pilot studies in areas not covered by other station projects and also for limited investigations which are appropriate but not of the magnitude to justify a separate project. Ram Epididymitis Studies: These studies were designed to characterize the role of Chlamydia psittaci in ram epididymitis and ovine abortion. Five strains of C. psittaci and three strains of C. trachomatis were used for monoclonal antibody studies. Preliminary results indicate that the turkey ornithosis strain is serologically related to the strains causing enzootic abortion of ewes. Osteopetrosis Studies: Investigations are continuing designed to better characterize the pathogenesis of osteopetrosis in Montana purebred Angus calves. Results of present investigations suggest that a defect may exist in the function of osteoclasts and/or the makeup of chondro-osseous matrix material. Monoclonal antibodies to bovine osteoclasts will be produced in order to tag these cells in animals with osteopetrosis. Specialized matrix histochemical studies are being performed on bones from both normal and osteopetrotic calves. Clinical Pathology: The Clinical Pathology Lab established computerized records for data analysis. This can provide baseline information relating to nutritional and/or environmental circumstances if abnormal levels are present in animals in different geographic locations or under different nutritional or environmental circumstances.
<p>PROGRESS: 1984/01 TO 1984/09<br/>
Ram Epididymitis: These studies were designed to investigate reproductive problems in rams and to determine if there was a correlation between several diagnostic parameters studied. The results of these investigations revealed that: (1) the CF test is the best single test for Brucella ovis infected rams; (2) numerous WBC's in the semen is a strong indicator of Brucella infection; (3) less than half of the RE rams out of 145 rams tested in two Montana flocks had infections associated with Brucella and (4) the combination of palpation, C.F. serology, semen evaluation and culture is the best diagnostic battery to use for RE. Osteopetrosis: Investigations of osteopetrosis (OP) in Montana calves showed that all OP calves studied to date were stillborn or died shortly after birth, were 2 to 3 weeks premture, were of small body size, had bracygnathia inferior, impacted molar teeth, open cranial fontanelles, fragile bones that lacked marrow cavities. Radiographically bones were dense with parallel striae of delicate trabeculae filling metaphyseal and diaphyseal marrow spaces. Histologically medullary bone consisted of densely packed primary and secondary trabeculae with abundant matrix material. Cellular density (clasts/blasts) was moderate to diminished with evidence of intermittent activity. Bovine OP appeared to be inherited as an autosomal recessive trait.
<p>PROGRESS: 1983/01 TO 1983/12<br/>
This project is used for initial or pilot studies in areas not covered by other station projects and also for limited investigations which are appropriate but not of the magnitude to justify a separate project. Ram Epididymitis Studies: These investigations have revealed that: (1) Chlamydia psittaci can often be isolated from the epididymides of field cases of ram epididymitis; (2) Chlamydia appear to have a tropism for genital tissues and frequently co-exist with either Actinobacillus or Brucella ovis in the epididymides; (3) Actino bacillus sp. cannot be transmitted to young virgin rams by any route other than by direct injection into the epididymis; (4) virulence of chlamydia in embryonated eggs is proportional to ability of the microorganism to grow rapidly and in large numbers. Osteopetrosis Studies: A total of 25 cases of osteopetrosis have been confirmed and studied in detail during the past three years. A thorough study of histopathologic and radiographic lesions revealed a unique form of osteodysplasia in all affected calves. Five purebred Angus cows and one purebred Angus bull have been identified as known carriers of the mutant gene of osteopetrosis. These six animals have been purchased by the Veterinary Research Laboratory and are being used for breeding and experimental studies. Ongoing investigations are designed to better characterize the pathogenesis of this genetic disease of Angus cattle.
<p>PROGRESS: 1981/01 TO 1981/12<br/>
This project is used for initial or pilot studies in areas not covered by other station projects and also for limited investigations which are appropriate but not of the magnitude to justify a separate project. Brucellosis Studies: The bovine brucellosis study on vertical transmission from dam to offspring is continuing. Results still indicate a significant seroconversion from negative to positive titers during the last trimester of pregancy. Osteopetrosis Studies: One emerging congenital disease affecting calves in Montana is osteopetrosis. This disease affects primarily red and black Angus calves. Most of the affected calves are born prementurely and are dead or die shortly after birth. They are slightly smaller in size than normal, have a shortened lower jaw and impacted molar teeth. Perhaps the most noticeable feature is that the long bones of the legs break very easily. In fact, even normal physicl pressures resulting from the birth process may fracture bones of these affected calves. Research studies are underway to determine why specialized bone cells (osteoclasts) fail to perform their normal function in affected calves. In addition, studies are being directed at developing a biochemical (blood) test to identify adult carriers of this lethal genetic disease of calves.
<p>PROGRESS: 1980/01 TO 1980/12<br/>
This project is used for initial or pilot studies in areas not covered by other station projects and also for limited investigations which are appropriate but not of the magnitude to justify a separate project. Brucellosis Studies - The bovine brucellosis study on vertical transmission from dam to offspring is continuing. The brucellosis positive heifer gave birth to a second calf which was not infected as determined by serology and culture. Observations for vertical transmission which occurred in the case of this heifer's dam to her offspring are being made. This heifer has been bred, determined to be pregnant and biweekly blood samples are taken and tested for any positive reactions for Brucella abortus antibodies. At the present time no rise in titer has been observed. Observations will continue through gestation and following parturition. Osteopetrosis Studies - A pilot project is underway to characterize the pathogenesis of hereditary osteopetrosis in newborn calves in Montana. Recent reports from the Montana Diagnostic Laboratory Bureau suggest that osteopetrosis in purebred Angus calves is occurring with considerable frequency in some herds. The pilot study will define breeding histories, characterize representative lesions and investigate the role of the immune system in the pathogenesis of the disease.
<p>PROGRESS: 1979/01 TO 1979/12<br/>
This project is used for initial or pilot studies in areas not covered by other station projects and also for limited investigations which are appropriate but not of the magnitude to justify a separate project. Brucellosis Studies. The bovine brucellosis study on vertical transmission from dam to offspring is continuing. The brucellosis positive heifer gave birth to a second calf which was not infected as determined by serology and culture. Observations for vertical transmission which occurred in the case of this heifer's dam to her offspring are being made.
<p>PROGRESS: 1978/01 TO 1978/12<br/>
This project is used for initial or pilot studies in areas not covered by other station projects and also for limited investigations which are appropriate but not of the magnitude to justify a separate project. Brucellosis Studies. The bovine brucellosis study on vertical transmission from dam to offspring is continuing. The brucellosis positive heifer from the previous work is now pregnant for the second time. From the first pregnancy she gave birth to a brucellosis infected premature calf. Observations in regard to this pregnancy will be made on a similar basis to the first pregnancy. This dam continues to have a serum titer of 1:3200 or higher as determined by testing every 2 weeks. Clinical Pathology Studies. During the past year initiation of clinical pathological studies has taken place for the purpose of determining clinical chemistry and cellular hematology of neonatal and parasitic diseases of livestock. Detection of such parameters in these disease processes may provide descriptive values for identification of these diseases in the subclinical state or diagnostic features of a specific disease syndrome.
<p>PROGRESS: 1977/01 TO 1977/12<br/>
This project is used for initial or pilot studies in areas not covered by other station projects and also for limited investigations which are appropriate but not of the magnitude to justify a separate project. Brucellosis Studies - A clinical investigation of brucellosis in regard to vertical transmission (from dam to offspring) was carried out during the past 3 years. A heifer calf was born from a brucellosis positive dam. Brucella abortus (Biotype I) was isolated from the dam's milk after calving and during the early lactating period. The calf was departed from the dam at 7 months of age. The heifer calf was bred and conceived as a 2 year old. She carried the fetus for 8 months before giving birth to a weak, premature calf from which Brucella abortus (Biotype I) was isolated from several organs and body fluids. During the later part of the gestation period the heifer demonstrated a serum rise of brucella abortus titer to a level of 1:12,800 immediately prior to parturition.
<p>PROGRESS: 1976/01 TO 1976/12<br/>
Brucellosis Studies: A clinical investigation of brucellosis is continuing in regard to infection in the bovine and equine. Animals which are infected or carrying positive serum titers are being periodically sampled for serum titers and cultures to establish baseline date prior to investigating factors which mayalter this baseline data. Those factors that will be evaluated are stress, estrous, conception, parturition, antibiotic therapy and possibly others. Presently one (cow) of the two animals are pregnant and the other (heifer) is being bred. Titer studies are continuing as will culture studies at appropriatetimes. In addition a cooperative study with personnel from State and Federal regulatory agencies (Montana Board of Livestock and Animal and Plant Health Inspection Service) is investigating the use of steroids in a brucellosis positive animal (serum titer positive and culture negative) to determine if large doses of steroids can influence the culture results in such animals.
<p>PROGRESS: 1975/01 TO 1975/12<br/>
Studies were initiated in early 1975 to determine whether small native rodents and related mammals are involved in the transmission of trichinellosis, brucellosis or leptospirosis in the Yellowstone Park area. A total of 82 mammals (81 rodents and 1 insectivore) from the Lamar and Pelican drainages in the eastern section of the Park were live-trapped, transported to the V.R.L. andexamined for the presence of Trichinella spiralis tissue larvae & for antibodiesto Bracella 10 Leptospira serotypes. All animals were negative for the three target infections. Brucellosis Studies: A clinical investigation of brucellosis is abortus and infections. Brucellosis Studies: A clinical investigation of brucellosis is being carried out in regard to infection in the bovine and equine. Animals which are infected or carrying positive serum titers are being periodically sampled for serum titers and cultures to establish baseline data prior to investigating factors which may alter this baseline data. Those factors that will be evaluated are stress, estrous, conception, parturition, antibiotic therapy and possibly others. Coal Tar Toxicity in Cattle: Because ofa possible coal tar enamel poisoning in cattle a study was initiated to determine if a coal tar enamel produce used to coat gas pipelines was toxic in cattle. In the first trial the tar product was administered via stomach tube in daily dosages of 0.5 ml/kg, 2.0 mg/kg, 5.0 mg/kg and 10 mg/kg.