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Introduction: Lymphomas are among the most important and common malignant tumors in cats. Differentiating lymphomas from reactive lymphoid proliferations can be challenging, so additional tools such as clonality assessment by PCR are important in diagnosis finding. Several PCR assays have been developed to assess clonality in feline lymphomas. For T-cell lymphomas TRG (T-cell receptor gamma) genes are the preferred target whereas for B-cell lymphomas most primer sets target immunoglobulin heavy chain (IGH) genes. Here we compare commonly used diagnostic primer sets for the assessment of clonality in feline lymphomas under controlled conditions (i.e., identical sample set, PCR setup, amplicon detection system)., Methods: Formalin-fixed and paraffin-embedded samples from 31 feline T-cell lymphomas, 29 B-cell lymphomas, and 11 non-neoplastic controls were analyzed by PCR combined with capillary electrophoresis., Results and Discussion: We show that the combination of the primer sets published by Weiss et al. and Mochizuki et al. provided the best results for T-cell clonality, i.e., correctly assigns most populations as clonal or polyclonal. For B-cell clonality, the combination of the primer sets by Mochizuki et al. and Rout et al. gave the best results when omitting the Kde gene rearrangement due to its low specificity. This study rigorously evaluated various primer sets under uniform experimental conditions to improve accuracy of lymphoma diagnostic and provides a recommendation for achieving the highest diagnostic precision in lymphoma clonality analysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Weyrich, Hecht, Köhler, Herden and Henrich.)

Predicting the outward appearance of dogs via their DNA, also known as Canine DNA Phenotyping, is a young, emerging field of research in forensic genetics. The few previous studies published in this respect were restricted to the consecutive analysis of single DNA markers, a process that is time- and sample-consuming and therefore not a viable option for limited forensic specimens. Here, we report on the development and evaluation of a Massively Parallel Sequencing (MPS) based molecular genetic assay, the LASSIE MPS Panel. This panel aims to predict externally visible as well as skeletal traits, which include coat color, coat pattern, coat structure, tail morphology, skull shape, ear shape, eye color and body size from DNA using 44 genetic markers in a single molecular genetic assay. A biostatistical naïve Bayes classification approach was applied to identify the most informative marker combinations for predicting phenotypes. Overall, the predictive performance was characterized by a very high classification success for some of the trait categories, and high to moderate success for others. The performance of the developed predictive framework was further evaluated using blind samples from three randomly selected dog individuals, whose appearance was well predicted., Competing Interests: Declaration of Competing Interest The Agrigenomics Team at ThermoFisher Scientific (USA) supported the study by aiding primer design and providing primer pools., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The popularity of dogs as human companions explains why these pets regularly come into focus in forensic cases such as bite attacks or accidents. Canine evidence, e.g., dog hairs, can also act as a link between the victim and suspect in a crime case due to the close contact between dogs and their owners. In line with human DNA identification, dog individualization from crime scene evidence is mainly based on the analysis of short tandem repeat (STR) markers. However, when the DNA profile does not match a reference, additional information regarding the appearance of the dog may provide substantial intelligence value. Key features of the dog's appearance, such as the body size and coat colour are well-recognizable and easy to describe even to non-dog experts, including most investigating officers and eyewitnesses. Therefore, it is reasonable to complement eyewitnesses' testimonies with externally visible traits predicted from associated canine DNA samples. Here, the feasibility and suitability of canine DNA phenotyping is explored from scratch in the form of a proof of concept study. To predict the overall appearance of an unknown dog from its DNA as accurately as possible, the following six traits were chosen: (1) coat colour, (2) coat pattern, (3) coat structure, (4) body size, (5) ear shape, and (6) tail length. A total of 21 genetic markers known for high predicting values for these traits were selected from previously published datasets, comprising 15 SNPs and six INDELS. Three of them belonged to SINE insertions. The experiments were designed in three phases. In the first two stages, the performance of the markers was tested on DNA samples from dogs with well-documented physical characteristics from different breeds. The final blind test, including dogs with initially withheld appearance information, showed that the majority of the selected markers allowed to develop composite sketches, providing a realistic impression of the tested dogs. We regard this study as the first attempt to evaluate the possibilities and limitations of forensic canine DNA phenotyping.

Bcl-2, an anti-apoptotic protein, is commonly overexpressed in follicular lymphomas in humans. This is usually the result of a chromosomal translocation that transposes the Bcl-2 gene into the immunoglobulin gene locus. The immunohistochemical assessment of this overexpression can be used as a tool for the differentiation of follicular lymphoma and follicular hyperplasia. In cats, little information about the expression of Bcl-2 in follicular lymphoma exists. We investigated 18 follicular lymphomas histologically and immunohistochemically for the expression of Bcl-2, CD3, CD45R, and feline leukemia virus. Clonality was assessed by PCR for antigen receptor gene rearrangements. Although the histology resembled that of their human counterparts, diffuse expression of Bcl-2 within the follicles of the feline lymphomas, as seen in human cases, was not present. Only single cells within the follicles, comparable to the reactive controls, were positive for Bcl-2 expression. The mean survival time of 4.6 y confirmed the indolent character of the tumor. None of the clinical parameters assessed were statistically significant predictors of survival. Furthermore, a statistically significant difference in survival of animals with or without anti-neoplastic therapy was also not demonstrable.

Crime scene samples originating from domestic dogs such as hair, blood, or saliva can be probative as possible transfer evidence in human crime and in dog attack cases. In the majority of such cases canine DNA identification using short tandem repeat (STR) analysis is the method of choice, which demands, among others, a systematic survey of allele frequency data in the relevant dog populations. A set of 13 highly polymorphic canine STR markers was used to analyze samples of 1,184 dogs (including 967 purebred dogs) from the so-called DACH countries (Germany, Austria, Switzerland). This CaDNAP 13-STR panel has previously been validated for canine identification in a forensic context. Here, we present robust estimates of allele frequencies, which are essential to assess the weight of the evidence by estimating the probability of a matching DNA profile within the dog population under question, e.g. in the form of a random match probability (RMP). The geographical provenance of the tested dogs showed a negligible influence on the observed genotype variation. Therefore, we combined the STR data from all three countries into a single dog population sample (DPS). In contrast, pronounced genetic differentiation between dog breeds was found by principal component analysis and sub-structure analysis with the STRUCTURE software. These findings entailed the need to account for the effects of DPS breed composition on allele frequency estimates. A possible strategy, which was favored here, relies on collecting a DPS that is guided by the breed composition of the relevant dog population. In total, dogs from 166 different breeds were included in our DPS, 64 of them including at least 5 individuals (n = 771 dogs). Sampling reflected the abundance of breeds in the DACH countries with the following being the most common ones: German Shepherds (population frequency: 14.3%), Dachshunds (5.9%), Labrador Retrievers (3.9%), and Golden Retrievers (3.2%). The pedigree listing of the purebred dogs in our DPS ranked German Shepherds (DPS frequency 8.5%) first, followed by Labrador Retrievers (3.9%), Golden Retrievers (3%), and Dachshunds (2.5%). RMP values based on overall allele frequencies and accounting for substructure using F ST between breeds ranged between 10-13 and 10-14 and represent a conservative approach of RMP assessment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We tested a panel of 13 highly polymorphic canine short tandem repeat (STR) markers for dog breed assignment using 392 dog samples from the 23 most popular breeds in Austria, Germany, and Switzerland. This STR panel had originally been selected for canine identification. The dog breeds sampled in this study featured a population frequency ≥1% and accounted for nearly 57% of the entire pedigree dog population in these three countries. Breed selection was based on a survey comprising records for nearly 1.9 million purebred dogs belonging to more than 500 different breeds. To derive breed membership from STR genotypes, a range of algorithms were used. These methods included discriminant analysis of principal components (DAPC), STRUCTURE, GeneClass2, and the adegenet package for R. STRUCTURE analyses suggested 21 distinct genetic clusters. Differentiation between most breeds was clearly discernable. Fourteen of 23 breeds (61%) exhibited maximum mean cluster membership proportions of more than 0.70 with a highest value of 0.90 found for Cavalier King Charles Spaniels. Dogs of only 6 breeds (26%) failed to consistently show only one major cluster. The DAPC method yielded the best assignment results in all 23 declared breeds with 97.5% assignment success. The frequency-based assignment test also provided a high success rate of 87%. These results indicate the potential viability of dog breed prediction using a well-established and sensitive set of 13 canine STR markers intended for forensic routine use., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Analysis of clonality is gaining importance in diagnosing lymphomas in veterinary medicine. Usually, PCR for the analysis of antigen receptor rearrangement (PARR) is followed by electrophoretic separation of the PCR products. Aim of this study was to test the feasibility of HRM for the assessment of clonality in B-cell lymphomas of cats. High resolution melting analysis differentiates PCR products by their different melting point using the decrease in fluorescence of an intercalating dye during melting of the PCR product. Additionally, the method is easy to use with no post-PCR manipulation of the samples. Forty-seven feline B-cell lymphomas and 31 reactive lymphatic proliferations of cats were investigated by PARR followed either by capillary electrophoresis or an HRM assay. To objectify the interpretation of the HRM results a recently published mathematical approach was applied to the melting curve. To overcome discrepancies between the visual interpretation and the mathematical approach, the latter was modified to include testing of reproducibility and recognition of pseudoclonality. In 11 of 47 lymphoma cases clonal populations were detectable by HRM assay compared to 14 of 47 lymphomas in which clonal populations were detected by capillary electrophoresis assay. Neither of the methods showed a clonal pattern in any of the reactive samples. However, the HRM assay showed a unique pattern in cases of follicular lymphatic hyperplasia that had no corresponding pattern in capillary electrophoresis., Conclusion: The capillary electrophoresis assay could identify 3 lymphomas that were not detected by the HRM assay and is therefore regarded superior to the HRM assay. The comparison however, was hampered by the overall bad performance of the PARR, that might be the consequence of insufficient primer binding due to somatic hypermutation of the binding sites during antigen stimulated proliferation of the B lymphocytes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Feline large granular lymphocyte lymphomas are rare but very aggressive tumors with a poor prognosis. In this study, a cell line from an abdominal effusion of a cat with large granular lymphoma was characterized. Immunophenotype staining was positive for CD3 and CD45R, and negative for CD4, CD8, CD56, CD79α, BLA.36 and NK1. A TCR γ gene rearrangement was detectable by PARR. Neither FeLV antigen nor exogenous FeLV provirus could be detected. A chromosomal instability associated with a centrosome hyperamplification could also be determined. The cell line is able to lyse target cells without antigen presentation or interaction with antigen presenting cells. Therefore, these cells were classified as genetically instable non-MHC-restricted cytotoxic T cells with large granular lymphocyte morphology., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

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.

T-cell receptor γ alternate reading frame protein (TARP) is expressed by human prostate epithelial, prostate cancer, and mammary cancer cells, but is not found in normal mammary tissue. To date, this protein has only been described in humans. Additionally, no animal model has been established to investigate the potential merits of TARP as tumor marker or a target for adoptive tumor immunotherapy. In this study conducted to characterize feline T-cell receptor γ sequences, constructs very similar to human TARP transcripts were obtained by RACE from the spleen and prostate gland of cats. Transcription of TARP in normal, hyperplastic, and neoplastic feline mammary tissues was evaluated by conventional RT-PCR. In felines similarly to the situation reported in humans, a C-region encoding two open reading frames is spliced to a J-region gene. In contrast to humans, the feline J-region gene was found to be a pseudogene containing a deletion within its recombination signal sequence. Our findings demonstrated that the feline TARP ortholog is transcribed in the prostate gland and mammary tumors but not normal mammary tissues as is the case with human TARP.

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.

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.

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., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

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., (2010 Elsevier B.V. All rights reserved.)

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., (Copyright 2010 Elsevier B.V. All rights reserved.)

The variable domains of antigen receptors are very diverse and assembled in a modular system from a number of V-, D-, and J-region genes. Here we describe additional variants of V- and J-region genes of the feline T-cell receptor gamma (TRG) as well as the corresponding RSSs retrieved from Trace Archive of feline genomic sequences. Additionally, an unusually recombined TRGV-domain containing a partial inverted repeat of the included J-region and a short interspersed element of the CAN-SINE family located within the feline T-cell receptor gamma locus are also described.

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.

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., ((c) 2009 S. Karger AG, Basel.)

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.

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.

European mouflon are in the focus of research since they were brought from the Tyrrhenic islands to the European mainland a hundred years ago. From the beginning many populations on European mainland suffer from different claw diseases which are unknown in their original habitats. Foot rot, the ovine purulent laminitis, whose existence im wild ruminants was negotiated some years before, furthermore claw alterations caused by primary or secondary lack of trace elements similar to the copper deficiency syndrome of the boreal deer species moose and reindeer and finally horn hyperplasia with a genetic background are found as main claw diseases in Central Europe. Object of this study was the acquiring of clinical parameters from blood for the installation of a mouflon-specific diagnostic profile "claw diseases". Count of leucocytes (WBC), activity of Alkaline phosphatase, serum contents of phosphorus, iron, copper and zinc were found to be parameters for successful differential diagnosis and control of progress in cure programs.

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., (Copyright 1999 W.B. Saunders Company Ltd.)

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.

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.

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.

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.

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.

Ingestion of food can result in an acute decline of serum thyrotropin (TSH) concentrations, but it is not known whether meal composition and/or stomach distension are influential. Normal men and women were given a normocaloric or hypocaloric, isobulk meal at lunch and at dinner in a randomized design. The normocaloric, but not the isobulk, meal resulted in a significant decline in serum TSH at both lunch and dinner; thyroid hormones and cortisol were not affected significantly. These findings suggest that meal composition is influential in the acute postprandial decline of serum TSH in man. A possible mechanism is food-induced elevation of somatostatin and consequent suppression of TSH secretion.

Based on 200 litters descending from 50 sows relations between MHS-genotype and reproductivity were investigated. Breeds used were "Deutsches Edelschwein", German Landrace and a crossbred line. Evaluation of MHS-status was done by Polymerase Chain Reaction. Reproductivity traits were piglet numbers at different ages and litter growth. The sows were not preselected in regard to the halothane gene. MHS-negative sows showed increased reproductivity towards MHS-positives and within the negatives, the homozygote sows were superior to the heterozygotes.

The karyotype of Ateles geoffroyi (Cebidae; Primates; Mammalia) was studied using G- and C-banding techniques, and standardized idiograms are proposed. A polymorphism of chromosome 3 due to a pericentric inversion has been identified for the first time. Genetic analysis of this polymorphism showed Mendelian inheritance.

Restriction endonuclease fragment patterns of mitochondrial DNA (mtDNA) in sheep were analysed with 11 enzymes. Four breeds (Merinolandschaf, Rhoenschaf, Schwarzkoepfiges Fleischschaf and Skudde) of domestic sheep and European Mouflon were examined. A restriction map with 28 cleavage sites of seven enzymes was established. KpnI and PstI do not cut ovine mtDNA. Two EcoRI fragments of Merinolandschaf, Rhoenschaf and Mouflon each were cloned and partially sequenced. Intraspecific nucleotide sequence differences within 1.101 kb ranged from 0.09 to 0.27%. Hybridization analysis with a fragment of porcine mtDNA along with sequencing data from cloned fragments was used for orientation of the restriction map along the bovine sequence. Ovine mtDNA sequences encompassing parts of the Cyt.b-, ND5-, CoIII- and ATPase6 genes were compared with the corresponding sequences of the bovine mtDNA.

A region of 50 kb around the human PTH gene was cloned and mapped by restriction analysis. Sequence analysis was performed and 3270bp determined, completing the sequence of the gene. The nucleotide sequence was analysed with regard to homology between human, bovine and rat PTH genes, and various potential cis-acting regulatory elements were identified. The gene region lacks an obvious CpG island. The PTH gene region in patients suffering from (pseudo)-hypoparathyroidism was investigated by Southern blotting. No detectable alteration in the fragment patterns was observed. Results of segregation analysis in families with affected individuals was inconclusive.

In a 7 months old foal with a male pseudohermaphroditism the cytogenetic investigation revealed a XO/XYY-mosaic with a centric fusion of the Y-chromosomes.

The nucleotide sequence of a 441-base cDNA encoding the bovine protamine has been determined. This insert, isolated from a bovine spermatid-specific cDNA library, encodes a polypeptide of 50 amino acids of which 26 are arginine, 7 are cysteine, and 2 are tyrosine. The insert contains the complete 3'-noncoding region of 150 bases and most of the 5'-noncoding region. The predicted amino-acid sequence of bovine protamine is about 96% homologous to ram protamine, 76% to boar protamine, 64% to mouse protamine 1 and 52% to human protamine 1 and contains the central, highly basic domain of four arginine clusters found in the trout protamines. Our results show that bovine protamine is 50 amino-acid residues in length and not 47 residues as previously published (Coelingh, J.P. et al. (1972) Biochim. Biophys. Acta 285, 1-14).