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Miscellaneous Investigations in Veterinary Molecular Biology

Investigators
Quinn, Mark
Institutions
Montana State University
Start date
2006
End date
2011
Objective
The short and long term objectives of research in VMB are the development of new drugs, vaccines, and diagnostic tools for fighting infectious diseases of livestock. This includes scholarly discovery and dissemination of science and technology related to diseases affecting livestock and wildlife, as well as zoonotic diseases that can be transmitted to humans.
More information
NON-TECHNICAL SUMMARY: Infectious disease causes considerable loss for livestock producers by reducing production of animal units and by reduced sales because of food safety concerns. Veterinary Molecular Biology (VMB) is the only research unit in Montana focused on animal health, particularly on the study of infectious diseases of cattle. New faculty members joining VMB 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 VMB. 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.

APPROACH: VMB 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.

PROGRESS: 2007/01 TO 2007/12
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.

IMPACT: 2007/01 TO 2007/12
All of these seed projects focus on infectious diseases important to Montana and the Nation. The impact is high, and it is hoped that these projects will lead to a better understanding of these diseases.

PROGRESS: 2006/01/01 TO 2006/12/31
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.

IMPACT: 2006/01/01 TO 2006/12/31
All of these seed projects focus on infectious diseases important to Montana and the Nation. The impact is high, and it is hoped that these projects will lead to a better understanding of these diseases.

PROGRESS: 2005/01/01 TO 2005/12/31
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).

IMPACT: 2005/01/01 TO 2005/12/31
Strangles remains one of the most significant infections of horses. The vaccines currently available suffer from both a lack of efficacy and at best, short term protection. Development of a long term, highly effective vaccine for strangles would make a significant contribution to the improvement of equine infectious disease management. Cross-comparison of herpes simplex virus 1 (HSV-1) with Marek's Disease virus and bovine herpesvirus 1 suggests that herpesviral latency results from the capacity of these viruses to exploit the host interferon response as a means to introduce ON-OFF switches into the viral life cycle. Potentially, elimination of viral interferon antagonists would be a simple and universal strategy to attenuate any herpesvirus, and could be used to produce safe and effective live vaccines against Marek's Disease and infectious bovine rhinotracheitis.

PROGRESS: 2004/01/01 TO 2004/12/31
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.

IMPACT: 2004/01/01 TO 2004/12/31
Strangles remains one of the most significant infections of horses. The vaccines currently available suffer from both a lack of efficacy and at best, short term protection. Development of a long term, highly effective vaccine for strangles would make a significant contribution to the improvement of equine infectious disease management. The deer and elk hunting industry contributes hundreds of millions of dollars each year to Montana economy. Chronic Wasting Disease has the potential to eliminate the industry. A live animal diagnostic test would make management of the spread of this disease much more practical then it is now.

PROGRESS: 2003/01/01 TO 2003/12/31
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.

IMPACT: 2003/01/01 TO 2003/12/31
Strangles remains one of the most significant infections of horses. The vaccines currently available suffer from both a lack of efficacy and at best, short term protection. Development of a long term, highly effective vaccine for strangles would make a significant contribution to the improvement of equine infectious disease management. The deer and elk hunting industry contributes hundreds of millions of dollars each year to Montana economy. Chronic Wasting Disease has the potential to eliminate the industry. A live animal diagnostic test would make management of the spread of this disease much more practical then it is now.

PROGRESS: 2002/01/01 TO 2002/12/31
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.

IMPACT: 2002/01/01 TO 2002/12/31
Shipping fever in calves accounts for hundreds of milliions of lost dollars annually in the cattle industry. This syndrome is caused by a number of viruses which then predispose the infected calves to secondary bacterial infection. This project will generate data that will increase our understanding of defense mechanism of calves to these pathogens. This information in turn can be used for the rational development of immunotherapies to these pathogens.

PROGRESS: 2001/01/01 TO 2001/12/31
(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.

IMPACT: 2001/01/01 TO 2001/12/31
(Harmsen) Bovine respiratory disease complex (shipping fever) is a group of economically important diseases of cattle and results in losses to the cattle industry of millions of dollars per year. The etiopathogenesis of shipping fever remains unknown. Our studies are focused on understanding the role of innate immune cells responding to the invasion by bacterial and viral organisms relevant to shipping fever. This understanding will strengthen the management efforts against shipping fever, which to date, have been ineffective in preventing and treating this disease complex. (Schmidt) Our goal is to measure how critical it is to use isogenic vectors for targeting mutations into bovine cells. Were targeted mutagenesis to become tractable in cattle, it could both increase the value of existing cattle-based commodities and allow creation of countless more valuable cattle-based products. Thus, it could allow production of particularly disease resistant herds or herds exhibiting increased milk or beef production. It could also allow cattle to be used for efficient production of protein-based pharmaceutical products, such as human insulin, human growth hormone, human blood clotting factors, and others.

PROGRESS: 2000/01/01 TO 2000/12/31
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.

IMPACT: 2000/01/01 TO 2000/12/31
The production of recombinant human proteins in livestock is likely to become a completely new multi-billion dollar branch of agriculture in the near future. The current proposal is aimed at improving the technology by which these procedures are performed. Importantly, this work will ensure that as this new branch of agricultural production develops, we will be at the forefront of the technology and will be able to make a major contribution to this new industry.

PROGRESS: 1999/01/01 TO 1999/12/31
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.

IMPACT: 1999/01/01 TO 1999/12/31
S. neurona causes equine protozoal myeloenchaphalitis(EPM),an economically important disease in horses. Over 50% of U.S. horses are infected with S. neurona and many develop neurological disorders. This research should eventually contribute the development of a vaccine against EPM. Rotavirus is the major viral cause of calf scours and cost the industry $500 million/year. Current vaccines fail to effectively control infections. We focus on the functions of viral protein NSP1 that may be responsible for toxic effects in cells.

PROGRESS: 1998/01/01 TO 1998/12/31
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.

PROGRESS: 1997/01/01 TO 1997/12/31
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.

PROGRESS: 1996/01 TO 1996/12
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.

PROGRESS: 1995/01 TO 1995/12
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.

PROGRESS: 1994/01 TO 1994/12
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.

PROGRESS: 1993/01 TO 1993/12
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.

PROGRESS: 1992/01 TO 1992/12
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).

PROGRESS: 1990/10 TO 1991/09
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.

PROGRESS: 1990/01 TO 1990/12
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.

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.

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.

PROGRESS: 1988/01 TO 1988/12
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.

PROGRESS: 1988/01 TO 1988/12
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.
PROGRESS: 1987/01 TO 1987/12

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.

PROGRESS: 1986/01 TO 1986/12
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.

PROGRESS: 1984/10 TO 1985/09
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.

PROGRESS: 1984/01 TO 1984/09
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.

PROGRESS: 1983/01 TO 1983/12
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.

PROGRESS: 1981/01 TO 1981/12
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.

PROGRESS: 1980/01 TO 1980/12
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.
PROGRESS: 1979/01 TO 1979/12
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.

PROGRESS: 1978/01 TO 1978/12
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.

PROGRESS: 1977/01 TO 1977/12
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.

PROGRESS: 1976/01 TO 1976/12
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.

PROGRESS: 1975/01 TO 1975/12
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 indaily dosages of 0.5 ml/kg, 2.0 mg/kg, 5.0 mg/kg and 10 ,g/kg. In the second trial it was administered via rumen fistula at a level of 1.0 to

PROGRESS: 1974/01 TO 1974/12
TRICHINOSIS STUDIES-prevalence of Trichinella spiralis larvae in tissues of 13 species carnivores was: Grizzly bear, 70%; lback bear, 0%; mountain lion, 62.1%;lynx, 100%; bobcat, 0%; river otter, 0%; wolverine, 0%; fisher, 100%; badger, 0%; skunk, 0%; timber wolf, 0%; coyote, 0%; raccoon, 0%. Equine Parsite Studies- acquisition of Parascaris equorum and subsequent course of infection in horsesstudied by periodic fecal examinations of 5 foals beginning at approximately 1 week of age. Patent infections developed in all 5 animals between 81 and 97 days afterinitial contact with pastures. Fecal egg counts reached initial peak within 2-6 weeks, followed occasionally by second, lesser peak. Anthelmintic Studies - field trial in which alfalfa pellets containing levamisole hydrochloride were creep-fed to calves undergoing exposure to lungworms indicated that a substantial disease occurred in number infected calves following initial administration medicated feed. However, weight gains did not differ significantly between treated and control calves at end of 132-day test period. Bovine Lungworm Studies - Post-mortem examination lungs from slaughter-age cattle originating from upper Yellowstone Valley Park County, Montana, indicated that cattle of this age do not ordinarily act as carriers of Dictyocaulus viviparus. No positive individuals found among 105 animals examined between January and June, 1974.

PROGRESS: 1973/01 TO 1973/12
Anaplasmosis Studies - Three of 6 pronghorn antelope collected in Rosebud Co., Montana in April and May, 1973 were serologically positive for anaplasmosis by the complement fixation test. Neither of 2 bovine calves which were inoculated with blood from these antelope developed titers for anaplasmosis during a 90-98 day post-inoculation period. Anthelmintic Studies - Levamisole hydrochloride forumated on a carrier of alfalfa pellets was fed twice via creep feeders to approximately 46 Hereford calves grazing on mountain summer range in Park County. Forty-two control calves in an adjacent pasture received nonmedicated pellets. The initial feeding of the drug resulted in an 84% reduction in the passage of lungworm. T (Dictyocaulus viviparus) larvae. The second feeding wasnot consumed uniformly by calves and no additional reduction occurred in output of lungworm larvae in feces. Rate of weight gain during the 125-day observationperiod was 1.77 lbs./day in treated calves and 1.84 lbs./day in controls. Bovine Ostertagiasis Studies - Studies on the prevalence of Ostertaglia spp. andtheir association with abomasal lesions in cattle from Gallatin County indicatedthat nematodes were present in the abomasum of all 51 animals examined post-mortem. The predominant species was O. ostertagi, with lesser numbers of O. bisonis and O. lyrata. Mean worm burden was 2,283 nemtodes, of which 18.6% were immature forms. Fecal egg counts averaged 36 eggs/gram; mean abomasal pH was 3.71. Parasite nodules were found in the abomasum of all animals examined, with independent nodules being the most common type.

PROGRESS: 1972/01 TO 1972/12
Anaplasmosis Studies - Seventeen of 20 pronghorn antelope collected in Rosebud Co., Montana between April and December, 1972 were serologically positive for anaplasmosis by the complement fixation test. However, none of 5 bovine calves which were inoculated with 50 ml. of blood from these antelope developed positive titers for anaplasmosis during a 3-month post-inoculation period. Trichinosis Studies - Studies on the prevalence and distribution of Trichinella spiralis in wild carnivorous mammals indicated that 10 species of native mammalswere infected, with trichina concentrations in muscle ranging from 0.01 to 708 larvae/gm. of tissue. The prevalence of T. spiralis among 371 individuals varied as follows: grizzly bear, 58%; mountain lion, 55%; wolverine 50%; fisher, 40%; coyote, 25%; bobcat, 17%; striped skunk, 17%; black bear, 12%; marten, 8%; and red fox, 7%. Anthelmintic Studies- Five calves given morantel tartrate in the form of medicated alfalfa pellets at the rate of 0.1 lb./100 lb.of body weight were completely cleared of Nematodirus helvetianus and Cooperia oncophora. Worm burdens in nontreated control calves averaged 3282 N. helvetianus and 2306 C. oncophora per animal. Clinical type projects of an applied nature are also included in this project. Comparison of titers in calves developed as the result of inoculation with an antigen at different timesin relation to weaning is in process at this time. Serums in all groups of calves have been collected and are being processed. In addition to the above this project is used for pilot or innovative investigations on a small scale to evaluate possible areas which may be worthy of later expanded consideration.

PROGRESS: 1971/01 TO 1971/12
In order to determine whether antibody production in response to vaccination will be influenced by the time the vaccination is given in relation to weaning, feedlot calves were divided into several groups. Each group was injected with aspecific antigen at a different time in relation to the weaning time, except forthe controls which were not vaccinated. The antigen used was a Vibrio fetus bacterin prepared in this laboratory. The techniques for determining antibodiesproduced by this vaccine is a system with which we are familiar, which is the reason for choosing this antigen. We hope to determine whether or not there areany significant differences between the levels of antibodies produced in these different groups of calves.

PROGRESS: 1970/01 TO 1970/12
Investigation continued on possible Mycoplasma and Chlamydial agents that were suspected as causes of abortions or birth of weak calves in Western Montana. Field isolates appeared to be non-pathogenic when inoculated into pregnant heifers and ewes. This preliminary work will continue as a formal project.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
View this project
Project number
MONB00022
Accession number
56879
Categories
Natural Toxins
Parasites
Bacterial Pathogens
Viruses and Prions
Commodities
Meat, Poultry, Game