DEFECTS IN MAMMALIAN SEXUAL DEVELOPMENT

• Sponsoring Institution: Cooperating Schools of Veterinary Medicine
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0087014
• Project Start Date: Jan 1, 1981
• Project End Date: Dec 31, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
James A. Baker Institute For Animal Health

Non Technical Summary
The goal of this proposal is to test and validate a method to in silico screen cDNA library databases created by high coverage direct sequencing, and determine whether this method is equivalent to gene detection by real time PCR. In other words, this new method should accurately determine whether cDNA for specific genes is present in a sample. If this method is valid, it will provide databases that can be repeatedly in silico screened for expression of multiple genes within metabolic pathways, providing a long term resource for each laboratory to test hypotheses of gene function. Once validated, this method can be proposed to construct similar cDNA sequence databases from affected animals of specific models to test hypotheses of gene expression and molecular pathway disruption. Significance to the equine industry: By using this test panel, owners and breeders can pass desirable performance traits in carriers to the next generation, without producing affected horses, and eventually eliminate these mutations from Thoroughbreds and Standardbreds. This would be achieved by identifying carriers and breeding them only to tested noncarriers, and thus avoiding production of affected horses. Testing at each generation, and removing those carriers that do not have desirable performance traits, will reduce the frequency of the mutation in the breed to the extent that it can eventually be eliminated. Thus, the panel would be incorporated into horse breeding programs as a selection tool for breeding Thoroughbred and Standardbred horses with high performance traits. Furthermore by testing young horses, owners can avoid unknowing investment in a young horse that has no breeding value. We anticipate the final panel will be available at Cornell University, readily accessible to the New York state racing industry, and will require either blood or hair samples for DNA testing.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3810	1040	10%
301	3810	1080	10%
301	3830	1040	10%
301	3830	1050	10%
301	3830	1080	10%
303	3810	1040	10%
303	3810	1080	10%
303	3830	1040	10%
303	3830	1050	10%
303	3830	1080	10%

Knowledge Area
303 - Genetic Improvement of Animals; 301 - Reproductive Performance of Animals;

Subject Of Investigation
3830 - Pets (companion animals); 3810 - Horses, ponies, and mules;

Field Of Science
1040 - Molecular biology; 1080 - Genetics; 1050 - Developmental biology;

Keywords

dog

horse

sex determination

infertility

sex differentiation

testis

disorders of sexual development

Goals / Objectives
The long term goal of this project is to produce expression databases from rare canine tissue that can be in silico screened multiple times. Our hypothesis is that cDNA libraries, directly sequenced with coverage >100x, when in silico screened in a sequence database, will reliably detect gene expression at the exon and cDNA transcript levels. Specific aims of this proposal are to: 1) Construct cDNA libraries from precisely timed canine embryonic and neonatal tissue, 2) Directly sequence libraries by a high coverage method, align sequences to the reference canine genome and prepare cDNA databases, 3) Identify gene expression by in silico screening of the databases (genome viewer, Excel files), and 4) Validate the method by comparison to real time PCR, in which expression is generated from original cDNA aliquots. At the end of this grant period, each investigator will have screened (in silico and real time PCR) rare tissue for genes of interest, providing preliminary gene expression results for external grant applications. A validated in silico screening method will allow detection of multiple gene expression in molecular pathways pertinent to molecular pathogenesis. Alternatively, if this new method is invalid, each investigator will have gained preliminary expression data on a sample of interest (real time PCR) and a frozen tissue bank resource (rare tissue samples at several developmental stages). By either outcome, this project will provide preliminary data and research resources needed by each investigator for external grant applications. Disorders of equine sexual development, such as cryptorchidism, testicular feminization, XX and XY sex reversal, and uterus masculinus are together, the major cause of inherited sterility or infertility reported in horses. Our specific aims are: 1) Develop a validated DNA-based test panel to screen cases of inherited equine sterility and infertility. This panel will screen the equine counterpart of genes known to cause cryptorchidism, testicular feminization, XY sex reversal, SRY-negative XX sex reversal and uterus masculinus in humans and other mammals. 2) Using the panel developed in Aim 1, we will screen a set of horses affected with SRY-negative XX sex reversal to test our hypothesis, identify causative gene mutations, and illustrate the utility of the panel. To develop the molecular testing panel, we will use the horse genome sequence to identify equine counterparts of 15 genes known to cause disorders of sexual development in humans and other mammals. For each gene, we will design primers, which are short DNA molecules used to generate multiple copies of a gene region during polymerase chain reaction (PCR). The primers will be validated by amplifying genes from a normal horse, sequencing the products, and comparing them to the Horse Genome sequence. To illustrate the utility of this panel, we will test six horses affected with SRY-negative XX sex reversal to identify causative mutations.

Project Methods
Canine samples collected at the appropriate stage for study are stored to preserve RNA and 1-2 per investigator (4-8 total) are processed for RNA extraction and cDNA production. Aliquots of cDNA are set aside for real time PCR and for cDNA library production, before fragmentation for direct sequencing. Libraries of cDNA are produced from an aliquot, then fragmented and directly sequenced by high coverage methods. Sequence output is processed, then aligned with the reference canine genome sequence. Housekeeping and candidate genes in each library, and their chromosomal locations, are identified by in silico screening (browser and Excel files). Aliquots of cDNA from each sample, previously set aside above, serve as templates in real time PCR to detect expression of the housekeeping and candidate genes/library. Results of each method are compared for significant differences by Chi Squared analysis. Using genes known to cause sterility and fertility in humans and other mammals, we will identify their equine counterparts in the horse genome database. We will design PCR primers, which are short DNA molecules used to generate (amplify) multiple copies of a portion of a gene during PCR. The primers will be validated by amplifying genes from a normal horse, sequencing the products, and comparing their nucleotide sequence to the gene sequence in the Horse Genome database. To illustrate the utility of this molecular diagnostic panel, genomic DNA from six horses affected with SRY-negative XX sex reversal will be similarly amplified by PCR and sequenced to identify causative mutations.

Progress 01/01/92 to 12/31/92

Outputs
Investigations continued to focus on the characterization of 'minute virus of canines' (canine parvovirus-1, MVC) and pathogenicity for pups and the developing fetus. Studies have clearly implicated MVC as a cause of reproductive failure in dogs infected during the early-mid stages of gestation (/`/25-40 days of pregnancy). Several clinical cases studied during the year have confirmed this virus as a natural cause of disease, but its general significance as a canine pathogen still remains uncertain. We have now developed DNA and RNA probes used in molecular hybridization analysis of MVC and is being used to examine tissues that failed to reveal MVC by conventional methods for viral genomes. We continue our work on canine brucellosis - principally in the development and assessment of diagnostic reagents. The improved slide agglutination test antigen using a mutant B. canis organism has proved the most sensitive and specific in extensive field evaluation in the U.S. and in Mexico. A third, and important, study has involved the collaborative development (with Colin Parrish et coll.) of an improved canine parvovirus type-2b vaccine. The improved vaccine, utilizing the most recent strain to emerge in the dog population, immunizes safely and efficiently. One of its advantages is the genetic stability of the attenuated virus after prolonged passage in cell culture. Collaborative studies with workers in Japan (A. Hashimoto, et. al.

Impacts
(N/A)

Publications

• MOWAT, G.N. ed (1992). Technological profile of applied vaccine production and quality control; biological standardization; constraints of vaccine production and quality control in developing countries (Carmichael). In: Report of the FAO.
• SELIKI, J.T., MIZAK, B., FLORE, H.P., GETTING, R.R., BURAND, J.P., CARMICHAEL, L. E., WOOD, H.A. and PARRISH, C.R. (1992). Canine parvovirus empty capsids produced by expression in a baculovirus vector: Use in analysis of viral properties.
• OKADA, Y., ISHIDA, K., HASHIMOTO, A., YAMAGUCHI, T., FUKUSHI, H., HIRAI, K. and CARMICHAEL, L.E. (1992) Virus reactivation in bitches with a history of canine herpesvirus. Am. Jour. Vet. Research. Accepted for publication (11/92).

Progress 01/01/91 to 12/30/91

Outputs
The following areas of investigation were addressed: 1) characterization of 'minute virus of canines' (canine parvovirus-1, MVC) and pathogenicity for pups and the developing fetus. Studies have further implicated MVC as a cause of reproductive failure in dogs. Experimental infections at different stages of gestation revealed that infection prior to gestational day 30 may cause fetal deaths and resorptions; infection at later times gave variable results that ranged from fetal myocarditis and hydrops fetalis to birth of pups that died shortly following delivery. Current studies focus on developing probes for in situ hybridization analysis of MVC pathogenesis in pregnant dams and in neonatal pups. MVC-DNA has been cloned into bacterial vector and a non-radioactive probe developed. Molecular characterization of MVC is partially completed. 2) A simple slide agglutination test for canine brucellosis has been improved to reduce non-specific (false-positive) reactions and extensively field tested. The improved antigen is much more specific, yet as sensitive, as the one currently available for canine brucellosis testing. 3) Collaborative studies have revealed high copy numbers of bovine warts virus DNA in equine sarcoids. Molecular characterization has been done and at least 2 different BPV DNA's were identified in sarcoid tissue. Results support the hypothesis that BPV, or a very similar virus, is linked to the cause of equine sarcoid.

Impacts
(N/A)

Publications

Progress 01/01/89 to 12/30/89

Outputs
The following areas of investigation were addressed this year: (1) Characterization of significant antigens of Brucella canis and their evaluation as diagnostic reagents; (2) Role of canine parvovirus type-1 (CPV-1, minute virus of canines-MCV) in canine reproductive disease. We have focussed this year on fetal infections and demonstrated that MVC can cause canine fetal deaths and abortion. The principal target organs appear to be the lung and small intestine. Work also continues on the molecular characterization of MVC and the development of DNA probes since viral isolation and other diagnostic methods have been unreliable with samples from field cases.

Impacts
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Publications

Progress 01/01/88 to 12/30/88

Outputs
We continue to explore methods for improved serodiagnostic tests for canine brucellosis. Several specific and cross-reactive antigens have been characterized and monoclonal antibodies have been raised against LPS determinants that are either specific for Brucella canis or which cross-react with other rough organisms. Work this year focused primarily on further characterization of cytoplasmic protein antigens and characterization of the most important ones by SDS-PAGE. Studies also have focused on the structural characterization of the minute virus of canines (MVC), a parvovirus whose pathogenicity is poorly defined. We continue to explore the role of the MVC in enteric illness of newborn pups and in its ability to cause reproductive disease in pregnant dogs. Recent isolation of the MVC from field cases of enteric illness in young pups leads us to believe that the MVC, which we have found to have a high seroprevalance in the dog population, may cause puppy illness that could be confused with infection by the highly pathogenic CPV-2. Further work is necessary to evaluate the pathogenicity of MVC for dogs and more sensitive/specific diagnostic procedures are needed. Monoclonal antibodies also have been raised against MVC that will be used in the development of diagnostic procedures and to explore the antigenic structure of this virus.

Impacts
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Publications

Progress 01/01/87 to 12/30/87

Outputs
A canine parvovirus type-2 variant (CPV-2a) that emerged around 1980 was found to possess greater virulence than the old CPV-2 which emerged in the dog population in 1980. In cooperation with scientists in Asia, Australia and Europe it was found that the variant is now the dominant type in the world dog population. It was determined that presently available vaccines (inactivated, experimental recombinant - baculovirus, empty capsid-, and live virus) protect against the CPV-2a. We continue to screen our Brucella canis gene library for antigenic proteins that may serve as specific diagnostic reagents. Current studies focus on defining the pathogenicity of the minute virus of canines for newborn pups and on the fetus. We have observed mild pathologic changes in exposed newborn pups, fetal deaths in inoculated pregnant dams and one instance of anasarca (hydrops fetalis), the latter also reported in humans infected during gestation with the human parvovirus B19. DNA isolated from equine sarcoids was found to be homologous with DNA from certain types of bovine papillomavirus (BPV). The role of BPV as the cause of equine sarcoid and breed susceptibility is under investigation.

Impacts
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Publications

Progress 01/01/86 to 12/30/86

Outputs
The kinetics of dog antibody responses to cytoplasmic protein antigens (CPA) from Brucella canis (B. canis) have been determined from the onset of infection through postinfection (pi) month 12. These have been determined by immunoprecipitation of radiolabeled antigens. Six or seven CPA provoke antibody responses that persist throughout the infectious period and two of them appear to prompt either "early" or "late" responses. We are currently establishing a B. canis gene library with the goal of cloning those genes that express desired CPA. Such proteins would have utility in the specific diagnosis of brucellosis in dogs and, possibly, in other species. We continue to study canine parvovirus (CPV) and other parvoviruses of carnivores with emphasis on analysis of the properties and evolution of the capsid protein gene. We are especially interested in determinants of host range (MU 64-73). One determinant of host range is the structure of the viral capsid. Other functions of that mapped to the capsid protein gene (MU 59-64) were the viral hemagglutinin. A non-hemagglutinating mutant of CPV also has been characterized. A mutant virus that appeared in 1981 (noted last CRIS report) continues to be isolated as the principal field type. We have found that the minute virus of canines replicates in lymphatic and intestinal tissues of pups. The infection was asymptomatic in young pups, but there was severe infection of the thymus and lymph nodes with marked degenerative changes in some pups.

Impacts
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Publications

Progress 01/01/85 to 12/30/85

Outputs
1. We have molecularly characterized five of the more than 27 B. canis cytoplasmic/periplasmic protein antigens by PAGE following immunoprecipitation. Certain proteins (eg. 66kD) are precipitated principally by antibodies in chronically infected dogs. Sera from dogs infected less than 3 months, however, contain antibodies that strongly precipitate a 31kD and >200 kK protein. Most sera also precipitated a 20 and 31 kD protein. There was considerable heterogeneity in the responses. Kinetic studies and further characterizations are in progress. Both B. canis specific and cross-reactive monoclonal antibodies have been prepared and characterized. 2. A mutant of canine parvovirus (CPV) derived after passage in feline cells that had altered host range (replicated in cat cells, but poorly in canine cells and in dogs) was characterized. Using genomic recombinations between native CPV and variant virus (CPV 102/10) and sequencing the DNA from regions that defined host range shift, differences in 2 AA in the capsid protein gene were noted. It appeared that a principal determinant of host range is the conformation of the viral capsid surface. 3. CPV strains appeared about 1981 that differed from the virus which originally emerged in 1978. The variant, distinguished by monoclonal antibodies, by restriction site differences and by DNA sequence analysis of "old" and "new" CPVs revealed that differences in two HphI sites are within the capsid protein gene.

Impacts
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Publications

Progress 01/01/84 to 12/30/84

Outputs
Work on B. canis has focused on the molecular characterization of diagnosticallysignificant cytoplasmic antigens (soluble antigens, SAs). Such antigens are highly conserved within the genus Brucella. Three protein/glycoprotein antigens have proved especially useful in diagnostic tests for B. canis. At least two "CSP" antigens (tabatabai, USDA, Ames, IA) extracted from the periplasmic space of B. abortus appear identical to the B. canis antigens. Also, a less mucoid (M-) variant of B. canis, found unsatisfactory as a vaccinal strain, has proved of value as antigen in agglutination tests. The M-organism differs in surface antigenic (LPS) properties from the M+(wild type) bacterium. Troublesome cross-reactions with non-Brucella organisms are greatly reduced. Antigenic variation within parvoviruses of canivores (dogs, cats, mink, raccoons) has been demonstrated with a panel of monoclonal antibodies. Analysis of variant parvoviruses by serological tests and competition radioimmune assays revealed that the capsid surface contained at least two determinants, each being comprised of different, but overlapping, epitopes. Genetic analysis of dog, cat and mink (MEV) parvoviruses by DNA recombination and sequence analysis of restriction fragments has revealed specific alterations in the parvoviral genome that related antigenic site variation to changes in vitro host range (dog vs. cat cells). Current work focuses on genetic analysis of structureal and functional changes in these parvoviruses.

Impacts
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Publications

Progress 01/01/83 to 12/30/83

Outputs
Brucella canis: Research focused principally on the partial purification of soluble (cytoplasmic) antigens (SAs), their characterization and utility as diagnostic antigens. Of the ca. 30 SAs revealed by PAGE, B. canis infected dogs commonly develop antibodies against 2 or 3 SAs; by PAGE followed by 'southern' blotting, 3 additional minor antigenic components are revealed in the sera of infected dogs. Tests of more than 100 field sera that had B. canis agglutinins by AGID or ELISA tests, using SAs, indicated good correlation with specific infection (culture-positve); no "false positive" sera reacted with the SAs, which are genus-specific (Brucella sp.). Canine parvovirus-2 (CPV-2): Biological (plaque, antigenic structure) and biochemical (DNA restriction enzyme, nucleotide sequencing of DNA regions coding for capsid antigens) markers have been identified for CPV-2 and, to a lesser extent, for selected feline, mink or raccoon parvoviruses, all which are closely related. Antigenic structures were compared and recombinant (CPV-FPV) viruses prepared and analyzed. A DNA region (ca. 60-65 map units) was identified that is associated with host range shift in cell culture. Variant CPVs have been partially characterized and markers identified that enable non-immunizing variants that emerge in vaccines to be recognized. Further characterization and analysis of CPVs, and other related parvoviruses of carnivores continue.

Impacts
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Publications

Progress 01/01/82 to 12/30/82

Outputs
Both cell wall and cytoplasmic antigens have been extracted from B. canis and characterized. Two strains were examined, one (M+) is virulent, the other (M-) a cell wall variant of reduced pathogenicity. Some extracted cell wall antigens have been purified and characterized. Three LPS antigens (2R, 3R, and 1R) were studied; one antigen (2R) had high B. canis specificity. It has been found useful as a diagnostic reagent for detecting B. canis infection, using AGID tests. More than 30 cytoplasmic antigens have been identified by PAGE. They are genus specific and may prove useful in developing more specific diagnostic tests for B. canis infection. One to four cytoplasmic antigens are revealed by AGID in sera from dogs of known infection status. An attenuated living canine parvovirus vaccine was developed and tested for safety and efficacy. Its biological and structural properties are being examined. Both biological and structural (DNA restriction enzyme profile and protein maps) differences have been revealed between virulent and attenuated virus. By conventional serology and use of a large battery of monoclonal antibodies (mAb) it has been possible to differentiate canine parvovirus (CPV) from feline panleukopenia virus (FPV). With the mAbs, however, various parvoviruses of carnivores (feline, dog, mink, raccoon) could readily be differentiated.

Impacts
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Publications

Progress 01/01/81 to 12/30/81

Outputs
Cell wall and internal antigens of Brucella canis were isolated and analyzed with respect to physico-chemical and serological characteristics. Certain antigens had broad reactivity with antigenic extracts from both Gram - and certain Gram + bacteria, others were specific for Brucella; one cell wall antigen reacted only with antisera from dogs infected with B. canis. Responses to the various antigenic extracts of B. canis were followed in experimentally infected beagles over a 5-year period. Strategies for immunizing dogs against canine parvovirus (CPV) were studied in laboratory and in field dogs. The role of maternal antibody in faillures to immunize was recognized and defined. A modified living canine parvoviral vaccine (Cornell strain 780916/'LP') was developed, field tested, and made available to commercial biologics firms after efficacy and safety studies had been satisfactorilly completed. Biologic and antigenic properties of CPV were defined and are being compared with those of antigenically-related parvoviruses from other species. Monoclonal antibodies (MAB) have been produced against CPV and the feline panleukopenia virus. One MAB against each virus was type-specific; another was type-common. Studies continue, with the objective of locating the antigenic determinants on the isolated viral proteins.

Impacts
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Publications