Serum anti-Müllerian hormone concentration as a diagnostic tool to identify testicular tissue in canine disorders of sexual development.

Disorders of sexual development (DSD) may have their origin in alterations of the chromosomal, gonadal or phenotypic sex. Affected animals are usually presented because of ambiguous external genitalia, seldom because of reproductive disorders. Anti-Müllerian hormone (AMH) is secreted in the gonads with higher amounts in males than in females and can be used to identify gonadal tissue in sexually normally developed dogs. The aim of this study was to examine the diagnostic potential of serum AMH to identify testicular tissue in 11 dogs with DSD. The diagnostic procedures applied were: determination of the phenotypic sex (n = 11), genital ultrasound (n = 9), determination of the SRY gene (n = 11), karyogram (n = 6), gonadectomy (n = 11), pathohistology of the gonads (n = 10), serum AMH measurement (n = 11). 39 female dogs described in a previous study and 19 male dogs with a normal spermiogram served as controls for the AMH serum concentrations in sexually intact dogs. The 11 dogs with DSD were classified as 7 XY DSD and 4 XX DSD. Presumptive testes were obtained in 10 dogs and 1 dog had an ovotestis combined with a testis. Mean serum AMH values of the dogs with DSD were significantly higher (P < 0.001) than in male and female controls. The upper limit of the AMH test (≥ 23ng/ml) was reached in 6 dogs. High AMH concentrations have been described previously in cryptorchid dogs. 1 dog with a male phenotype and 2 with a female phenotype had AMH values within the range of the male controls, although all of them had cryptorchid testes. A Poodle, in which epididymis were identified but no definitive gonads, had an AMH concentration of the lower limit of the test (≤ 0.01 ng/ml), comparable to previously described castrated dogs. This study indicates that serum AMH levels are a useful diagnostic tool to identify testicular tissue in dogs with DSD and suggests the possible use of AMH to diagnose testicular dysgenesis.

Neuronal ceroid lipofuscinosis (NCL) is caused by the entire deletion of CLN8 in the Alpenländische Dachsbracke dog.

Neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage diseases that have been described in a variety of dog breeds, where they are caused by different mutations in different genes. However, the causative gene defect in the breed Alpenländische Dachsbracke remained unknown so far. Here we present two confirmed cases of NCL in Alpenländische Dachsbracke dogs from different litters of the same sire with a different dam harboring the same underlying novel mutation in the CLN8 gene. Case 1, a 2-year-old male Alpenländische Dachsbracke was presented with neurological signs including disorientation, character changes including anxiety states and aggressiveness, sudden blindness and reduction of food intake. Magnetic resonance imaging (MRI) scans showed cerebral atrophy with dilation of all cerebral ventricles, thinning of the intermediate mass of the thalamus and widening of the cerebral sulci. Postmortem examination of the central nervous system (CNS) showed neuronal loss in the cerebral cortex, cerebellum and spinal cord with massive intracellular deposits of ceroid pigment. Additional ceroid-lipofuscin deposits were observed in the enteric nervous system and in macrophages within spleen, lymph nodes and lung. Ultrastructural analyses confirmed NCL with the presence of osmiophilic membrane bounded lamellar-like structures. Case 2, a 1,5-year old female Alpenländische Dachsbracke was presented with progressive generalized forebrain disease including mental changes such as fearful reactions to various kinds of external stimuli and disorientation. The dog also displayed seizures, absence of menace reactions and negative cotton-ball test with normal pupillary light reactions. The clinical and post mortem examination yielded similar results in the brain as in Case 1. Whole genome sequencing of Case 1 and PCR results of both cases revealed a homozygous deletion encompassing the entire CLN8 gene as the most likely causative mutation for the NCL form observed in both cases. The deletion follows recessive inheritance since the dam and a healthy male littermate of Case 1 were tested as heterozygous carriers. This is the first detailed description of CLN8 gene associated NCL in Alpenländische Dachsbracke dogs and thus provides a novel canine CLN8 model for this lysosomal storage disease. The presence of ceroid lipofuscin in extracerebral tissues may help to confirm the diagnosis of NCL in vivo, especially in new dog breeds where the underlying mutation is not known.

Validation of two canine STR multiplex-assays following the ISFG recommendations for non-human DNA analysis.

To gain general acceptance forensic DNA testing in animals needs to improve standardization of analysis methods and data interpretation. Recently, the International Society of Forensic Genetics (ISFG) took particular care of this topic by publishing recommendations for forensic non-human DNA analysis following the successful example of human DNA analysis in order to provide a basis for harmonization of the still existing inter-laboratory variability. By following these recommendations we demonstrate the performance of two short tandem repeat (STR) multiplexes for forensic identity testing of canine biological material. Thirteen STRs and two sex-specific markers were selected and validated according to the ISFG guidelines. Population genetic parameters were calculated based on 295 dog samples collected in Austria (124) and Germany (171). A repeat-based nomenclature of the mainly tetrameric STRs and corresponding allelic ladders are presented. All 146 different alleles included in the ladders were sequenced for correct allele calling. Additionally, a canine cell line (DH82-D3167) was evaluated as standard reference material.

Rubber jaw in a Weimaraner dog due to juvenile nephropathy. A case without evidence for genetic involvement.

The case of a 5-month-old male Weimaraner dog with severe osteodystrophia fibrosa (rubber jaw) and renal insufficiency is presented. Kidneys were smaller than normal with a granular cortical surface and a histopathological end-stage diagnosis consistent with juvenile nephropathy. Analysis of four described genetic mutations associated with Alport syndrome in dogs revealed no evidence for familiar inheritance in this dog.

Aprosencephaly with otocephaly in a lamb (Ovis aries).

Aprosencephaly is a rare condition in veterinary and human medicine characterized by the complete absence of telencephalon and diencephalon. Some cases are accompanied by a facial dysmorphism designated as otocephaly. A stillborn lamb had splanchnocranial anomalies that were classified by computed tomography, magnetic resonance imaging, and pathologic examination as aprosencephaly and otocephaly. The brain included parts of the cerebellum and brainstem but no telencephalon, diencephalon, or mesencephalon. The cerebellum had a structurally normal cortex with expression of neuronal nuclear antigen in the inner and doublecortin in the outer granular cell layers, as well as an irregularly situated nucleus dentatus. Aprosencephaly with otocephaly has been described in mice with heterozygous mutations in the Otx2 gene; however, no causative polymorphisms were detected in the Otx2 gene region of this lamb.

Cell lines derived from feline fibrosarcoma display unstable chromosomal aneuploidy and additionally centrosome number aberrations.

The purpose of the study was to evaluate clonality and presence of numerical chromosomal and centrosomal aberrations in 5 established feline fibrosarcoma cell lines and in a fetal dermal fibroblast cell line as a control. The clonality of all cell lines was examined using limited-dilution cloning. The number of chromosomes was counted in metaphase spreads. The immunocytochemical analysis of centrosome numbers was performed by indirect immunofluorescence using a monoclonal antibody that targets γ-tubulin, a well-characterized component of centrosomes. Monoclonal cell populations could be established from all cell lines. In all feline fibrosarcoma cell lines, the number of chromosomes deviated abnormally from the normal feline chromosome number of 2n = 38, ranging from 19 to 155 chromosomes per cell. Centrosome hyperamplification was observed in all 5 feline fibrosarcoma cell lines with a proportion of cells (5.7 to 15.2%) having more than 2 centrosomes. In the control cell line, only 0.6% of the cells had more than 2 centrosomes. In conclusion, the examinations revealed that centrosome hyperamplification occurs in feline fibrosarcoma cell lines. The feline fibrosarcoma cell lines possessed 10 to 25 times as many cells with centrosome hyperamplification as the control cell line. These observations suggest an association of numerical centrosome aberrations with karyotype instability by increasing the frequency of chromosome missegregation. The results of this study may be helpful for further characterization of feline fibrosarcomas and may contribute to the knowledge of cytogenetic factors that may be important for the pathogenesis of feline fibrosarcomas.

ISFG: recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations.

The use of non-human DNA typing in forensic science investigations, and specifically that from animal DNA, is ever increasing. The term animal DNA in this document refers to animal species encountered in a forensic science examination but does not include human DNA. Non-human DNA may either be: the trade and possession of a species, or products derived from a species, which is contrary to legislation; as evidence where the crime is against a person or property; instances of animal cruelty; or where the animal is the offender. The first instance is addressed by determining the species present, and the other scenarios can often be addressed by assigning a DNA sample to a particular individual organism. Currently there is little standardization of methodologies used in the forensic analysis of animal DNA or in reporting styles. The recommendations in this document relate specifically to animal DNA that is integral to a forensic science investigation and are not relevant to the breeding of animals for commercial purposes. This DNA commission was formed out of discussions at the International Society for Forensic Genetics 23rd Congress in Buenos Aires to outline recommendations on the use of non-human DNA in a forensic science investigation. Due to the scope of non-human DNA typing that is possible, the remit of this commission is confined to animal DNA typing only.

Disorders of sex development in the dog-Adoption of a new nomenclature and reclassification of reported cases.

Intersexuality is a rare congenital abnormality in domestic animals. It is reported in numerous species including the swine, goat, horse, cat, and dog. The present work provides an overview of the variety of intersexual conditions known in different dog breeds. Each case was reclassified based on the described gonadal constitution, reproductive tract abnormalities and karyogram, and categorised according to the stages normal sex development is undergoing resulting in three main categories: (1) sex chromosome disorders, (2) disorders of gonadal sex development, and (3) disorders of phenotypic sex development. Reclassification disclosed that the current classification scheme and terminology are inconsistently used in literature masking the real occurrence and frequency of various intersex conditions in dogs. For establishment of an individual, precise and definite diagnosis, introduction of a new nomenclature is proposed as recently recommended for humans. The new terminology is based on the gonosomal constellation and gonadal constitution, contributes to a systematic classification of canine intersex cases, and replaces the common but confusing diagnoses "true hermaphrodite" and "pseudohermaphrodite". The literature survey was supplemented by adding the results from own investigations in a German Pinscher and Berger Picard dog with bilateral ovotestes and ambiguous external genitalia. The diagnostic approach and clinical, pathomorphological and cytogenetic findings were described in detail.

High prevalence of non-productive FeLV infection in necropsied cats and significant association with pathological findings.

Applying a combination of semi-nested PCR and immunohistology (IHC), the presence of exogenous feline leukemia virus infection was studied in 302 necropsied cats with various disorders. 9% showed the classical outcome of persistent productive FeLV infection which was represented by FeLV antigen expression in different organs. 152 cats (50%) harboured exogenous FeLV-specific proviral sequences in the bone marrow but did not express viral antigen. These cats were considered as horizontally but non-productively infected. Statistical evaluation showed a significant association of non-productive horizontal FeLV infection with a variety of parameters. Non-productively infected cats were statistically significantly older and more often originated from animal shelters than cats without exogenous FeLV infection. Furthermore, some pathological disorders like anemia, panleukopenia, and purulent inflammation showed significant association with non-productive FeLV infection. No significant association was found with lymphosarcoma, known for a long time to be induced by productive FeLV infection.

Genetic characterization of a sheep-dwarf goat hybrid.

Twelve weeks after repeated spontaneous mating between a Bentheimer Landschaf ram and a West African dwarf doe was observed, the doe aborted a dead fetus. The aim of this study was to verify the parentage and the species of the supposed parents and the hybrid status of the fetus, using cytogenetic and molecular genetic methods. For this purpose, karyotypes were prepared using fresh blood samples from the ram and the doe, and genomic DNA was extracted from blood of the suspected parents and tissue of the aborted fetus. Fragments of the nuclear DNA-encoded interleukin-2 gene and the mitochondrial DNA-encoded 16S ribosomal RNA were sequenced and 19 microsatellites were genotyped in all 3 animals. The karyotypes and DNA sequences of the ram and the doe corresponded to domestic sheep and goat, respectively. The interleukin-2 sequence of the fetus was heterozygous at all positions where sheep and goat have different nucleotides. None of the 19 microsatellites excluded the ram and the dwarf doe as parents of the fetus. Taken together, we can conclude that the ram and the dwarf doe were from the species Ovis aries and Capra hircus, respectively, and that they were most likely the parents of the aborted fetus, which itself proved to be a hybrid of these 2 species.

A new subgroup of immunoglobulin heavy chain variable region genes for the assessment of clonality in feline B-cell lymphomas.

In human medicine, PCR-amplification of the complementarity determining region 3 of the immunoglobulin heavy chain genes followed by polyacrylamide gel electrophoresis (PAGE) is an accepted method to assess clonality in B-cell lymphomas and thereby facilitates the differentiation of neoplasias from benign hyperplasias or reactive infiltrates. To generate a basis for the development of a PCR-based assay for the assessment of clonality in feline B-cell lymphomas we analyzed 28 transcripts (cDNA) of the feline immunoglobulin heavy chain variable region genes (IGHV). Transcripts were generated using techniques for the amplification of unknown sequences (i.e. the SMART RACE and the CapFishing technique) as well as primers derived from sequences of the NCBI Trace archive of the cat. Analysis of this archive revealed traces similar to the human IGHV-1 and IGHV-3 subgroups of genes. By identification of the subgroup-specific leader sequence within the traces, two subgroup-specific primers for this region were designed and used to amplify the heavy chain variable region genes. Using all amplification techniques, transcripts of both subgroups were created and the subgroups were denominated according to their human counterparts as feline IGHV-1 and feline IGHV-3. By aligning previously described transcripts of the feline IGHV genes to our transcripts we were able to assign these to the IGHV-3 subgroup; therefore, this study provides the first description of the feline IGHV-1 subgroup of genes. On the basis of the IGHV-1 and IGHV-3 transcripts we developed a PCR-based assay. For each of the two subgroups we used one sense primer derived from the first and one sense primer derived from the third framework region each in combination with a mixture of three antisense primers derived from the fourth framework region. With these four sets of primers, the assay was able to detect monoclonality in 7/10 (70%) cats with histologically and immunohistochemically diagnosed B-cell lymphomas. In two of these cases, monoclonal rearrangement of the IGHV genes was only detectable with IGHV-1 subgroup-specific primers. Amplification of feline hyperplastic lymphatic tissue only gave results indicative of polyclonal populations. The use of a PCR-based assay in combination with standard techniques for the diagnosis of feline lymphoma is helpful and the characterization of the additional subgroup of feline variable regions genes puts this assay on a broader basis.

A complex malformation in a pig: case report and review of the literature.

Congenital defects like myofibrillar dysplasia (splayleg), umbilical and inguinal hernias, cryptorchism, intersexes, and anal atresia occur relatively frequently in swine. On the other hand, some developmental anomalies like double monsters are very rare. The present paper reports a rare case of a congenital complex malformation including polymelia, duplicitas coli partialis et recti, atresia ani et fistula rectogenitalis, duplicitas corpori uteri, cervicis, vaginae et vulvae and duplicitas vesicae, urethrae et renalis. A plausible interpretation concerning the etiology is that the anomalies arose from unequal partial twinning. The pig has been healthy and inconspicuous. Although no anus was formed defecation took place via a fistula to one of the vaginas. Posture and behaviour of the pig were normal. Cytogenetic analysis of blood lymphocytes revealed no numerical or gross structural anomalies. There have been no further piglets with developmental disorders in the same litter, in a second litter of the same parents and in other twelve litters by the same boar.

Characterization of C-, J- and V-region-genes of the feline T-cell receptor gamma.

Lymphomas and leukemias are important neoplasias of domestic cats and human beings. In some cases it can be difficult to differentiate these tumors from reactive lymphatic hyperplasia. To overcome this problem, the diagnosis of lymphomas and leukemias in man is often supported by molecular techniques. To be able to establish such a technique in the cat we had to sequence the genes coding for the antigen receptors. As primary target in this study we choose the T-cell receptor gamma. Using 5'-and 3'-RACE techniques we were able to clone and sequence four different V-region genes, which can be clustered into two subgroups as well as six variants of the C-region gene. Additionally, we found eight J-region genes which can be classified into three subgroups. One of the V-region genes, six of the J-region genes and all C-region genes had not been described previously. All together we analysed 112 clones containing V- and J-region genes and 31 clones containing C-region genes. Sixty-six of these clones were full length containing the L-region as well as the 5'-UTR of the feline T-cell receptor gamma. The sequences of the V-region- and J-region-genes show sufficiently homologous areas that can be used to establish a small number of consensus-primers to be applied in molecular diagnosis of feline lymphomas and leukemias.

Isolation and characterization of the porcine c-myc proto-oncogene and chromosomal assignment to SSC 4p13.

The proto-oncogene c-myc codes for a nuclear phosphoprotein, a transcription factor composed of the typical basic/helix-loop-helix/leucine zipper domains. Its expression is coupled to a multitude of physiological processes and regulated by a variety of hormones, growth factors, cytokines, lymphokines and the nutritional status, development and differentiation. Its key roles have been characterized, e.g. in adipogenesis, myogenesis and folliculogenesis. We have isolated and sequenced a 6.4-kb genomic fragment encoding the porcine c-myc proto-oncogene. The gene shows the typical c-myc structure with three exons, three putative promoters and a deduced protein of 439 amino acids. The porcine c-myc was mapped to chromosome 4p13 by screening of a porcine-rodent hybrid cell panel.

A diagnostic assay discriminating between two major Ovis aries mitochondrial DNA haplogroups.

Two major Ovis aries mitochondrial DNA (mtDNA) haplogroups have been described in independent studies. HinfI RFLP data of mitochondrial genomes from a large sample set (n = 239) indicated an ancient mutation which differentiates between the two mtDNA types. A completely determined sheep mtDNA sequence was used to assign this mutation to the COI gene and to develop a PCR based assay discriminating between the two phylogenetic branches. The haplogroup specificity of the mutation was further investigated in 26 randomly selected individuals. The animals were unequivocally assigned to their respective groups on the basis of the developed test and their complete control region sequences. The assay provides a rapid and economic means of discriminating between both major domestic sheep mtDNAs.

DNA content and ploidy level of bovine placentomal trophoblast giant cells.

Cytophotometric measurement of the DNA content of Feulgen-stained nuclei in touch preparations of bovine placentomes (n =5) revealed that 8C nuclei occurred in all, 16C nuclei in two, and 32C nuclei in one specimen. The determination of ploidy level by in situ hybridization with a Y-chromosome specific DNA probe showed that the majority of the fetal nuclei in touch preparations of placentomes from male fetuses (n =5) are tetraploid. Generally two tetraploid nuclei lie close together. These findings indicate that polyploidization is a normal feature in the development of the mostly binucleate trophoblast giant cells (TGCs). A new model for the development of these cells is proposed: a primary acytokinetic mitosis leads to a binucleate cell with two diploid nuclei. This cell enters a second acytokinetic mitosis during which the chromosomes of both nuclei form a common metaphase plate. The resulting cell with two tetraploid nuclei undergoes an additional S-phase but does not enter a renewed mitosis. The functional significance of this genome multiplication may be an increased synthetic capacity of bovine TGCs, caused by an increased number of gene copies available for transcription. Since genome multiplication is a property of invasive trophoblast cells of different species, it may be advantageous for trophoblast invasion.

The initial complication rate of phacoemulsification in India.

PURPOSE: This study was designed to investigate the feasibility of teaching experienced surgeons to perform phacoemulsification in India, a cataract-endemic area. Complications occurring during surgery and the first postoperative day were documented and evaluated. METHODS: During a 1-month period, at the Aravind Eye Hospital in Madurai, India, the first 100 consecutive cataract operations performed by each of three experienced surgeons (a total of 300 cases), using phacoemulsification were prospectively evaluated. Multiple logistic regression was used to identify factors associated with intraoperative and postoperative complications. RESULTS: The mean age of patients was 57.4+/-9.3 years. The median best corrected preoperative visual acuity was 20/80. Mean surgical and phacoemulsification times were 15.8+/-3.7 minutes and 2.2+/-1.5 minutes, respectively. Complications occurred in 65 (21.7%) eyes. The most common was a rent in the posterior capsule, occurring in 40 (13.3%) eyes. There were significant variations in complication rate and in surgical time among the surgeons. The risk of experiencing a complication decreased as the number of phacoemulsifications performed increased. An increased risk of complications was associated with worse preoperative visual acuity and increasing patient age. CONCLUSIONS: With each successive case, the chances of experiencing a complication decreased 1%. Acceptable results were obtained within 1 month of performing the first phacoemulsification.

Conserved Y-chromosomal location of TSPY in Bovidae.

We determined the chromosomal location of TSPY, the testis-specific protein, Y-encoded, by fluorescence in situ hybridization (FISH) to chromosome spreads of cattle, goat and sheep. Using a cloned polymerase chain reaction (PCR) product of one bovine TSPY family member, we were able to show a conserved Y chromosomal localization for TSPY in all three species. In contrast to a limited regional distribution of TSPY FISH signals on the chromosome of man, other primates, great apes, goat and sheep, in cattle TSPY-related sequences appear to be spread over most of the Y chromosome. The painting effect observed in this species reflects the higher complexity of the bovine TSPY gene family, being composed not only of a tandemly repeated cluster, but harbouring a large number of different family members dispersed all over the Y chromosome.

[Comparison of different methods for the diagnosis of freemartinism--blood group serology, cytology and polymerase chain reaction]. / Vergleich verschiedener Methoden zur Diagnose des bovinen Freemartinismus--Blutgruppenserologie, Zytologie und Polymerase-Ketten-Reaktion.

The percentage of freemartins among blood samples tested by chromosome analysis amounted to 83.9%, by blood group serologie 71.4%. 126 blood samples have been tested by blood group serology and PCR. Employing blood group serology, 71.3% and using PCR with BOV97M primers 85.8% of the animals proved to be freemartins. 40 blood samples were additionally analysed using PCR with zinc-finger-gene primers. 36 animals (90%) were identified as being freemartins by means of BOV97M and 34 animals (85%) by means of the zinc-finger-gene primer. The PCR method proved to be a rapid and very sensitive method for the diagnosis of freemartins and also suitable for routine testing. The BOV97M primer showed to have a higher Y chromosome specificity than the zinc-finger-gene primer.