p53 Gene Mutation in Blood of Zebu Cattle with Squamous Cell Carcinoma of the Horn.

Horn cancer is one of the most important diseases in Zebu castrated male cattle. The objective of this study was to investigate the presence of p53 gene mutation in the blood of affected cattle and its value for early diagnosis and prognosis. The study was conducted on blood samples from 20 affected cattle and six healthy control cattle from Western India. Plasma samples were evaluated for the presence of p53 gene mutation using the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique and the results were correlated with the stage of cancer. Five of the 20 cases had stage I neoplasms, nine stage II and six stage III, based on histopathological examination. PCR-SSCP analysis revealed an aberrant pattern of DNA migration on polyacrylamide gel electrophoresis of DNA extracts from blood samples of six animals with stage II and stage III cancer. No mutation was identified in blood from cattle with stage I cancer or from healthy control cattle. These results suggest that PCR-SSCP detection of p53 gene mutation in blood has potential diagnostic and prognostic value, and indicate the need for further large-scale investigation.

Molecular portrait of squamous cell carcinoma of the bovine horn evaluated by high-throughput targeted exome sequencing: a preliminary report.

BACKGROUND: Squamous Cell Carcinoma of horn, also known as horn cancer, is a prevailing type of cancer in cattles especially Bos indicus. It is one of the most prevalent disease in Indian bullocks often resulting in death and huge economic losses to farmers. Here, we have reported the use of targeted exome sequencing to identify variants present in horn cancer affected horn mucosa tissue and blood of the same animal to identify some of the prevalent markers of horn cancer. RESULTS: We have observed higher number of variants present in tissue as compared to blood as well as among cancer samples compared to samples from normal animals. Eighty six and 1437 cancer-specific variants were identified among the predicted variants in blood and tissue samples, respectively. Total 25 missense variants were observed distributed over 18 genes. KRT8 gene coding for Keratin8, one of the key constituents of horn, displayed 5 missense variants. Additionally, three other genes involved in apoptosis pathway and two genes involved in antigen presentation and processing also contained variants. CONCLUSIONS: Several genes involved in various apoptotic pathways were found to contain non-synonymous mutations. Keratin8 coding for Keratin, a chief constituent of horn was observed to have the highest number of mutations. In all, we present a preliminary report of mutations observed in horn cancer.

Carcinoma espinocelular da base do chifre, bilateral e simétrico, em vaca com distúrbio hormonal ˗ relato de caso / [Horn-based squamous cell carcinoma, bilateral and symmetrical in cow with hormonal disorder ˗ case report]

Este trabalho tem por objetivo descrever um caso de carcinoma espinocelular da base do chifre, bilateral e simétrico, em uma vaca e discutir a questão hormonal, possivelmente envolvida na patogênese da doença. Tratava-se de uma fêmea bovina, 11 anos, com histórico de emagrecimento progressivo, presença de massas exofíticas na base de ambos os chifres e em anestro por muitos anos. Foi realizada biopsia incisional de ambas as lesões para exame histopatológico e dosagens hormonais. Devido à progressão do quadro clínico, optou-se pela eutanásia, seguida dos exames necroscópico e histopatológico. O exame histopatológico revelou tratar-se de um carcinoma espinocelular infiltrativo bem diferenciado, e as dosagens hormonais apresentaram alterações nos níveis do hormônio luteinizante, folículo estimulante e estrógenos totais. Apesar de existirem descrições de carcinomas espinocelulares da base do chifre, ainda não havia relatos da ocorrência do mesmo bilateral e em uma vaca com distúrbios hormonais.(AU)

The objective of this study was to describe a case of bilateral and symmetrical squamous cell carcinoma from the horn base in a cow and to discuss the hormonal question, possibly involved in its pathogenesis. A 11-year-old beef cow presenting a history of progressive thinning, presence of exophytic masses at the base of both horns and anestrous for many years was assisted. An incisional biopsy of both lesions was performed for histopathological examination and hormonal dosages. Due to the clinical progression, euthanasia followed by necroscopic and histopathological examination was carried out. Histopathological examination revealed a well differentiated infiltrative squamous cell carcinoma and the hormonal dosages presented changes in luteinizing hormone, follicle stimulating and total estrogen levels. Although there are descriptions of basal squamous cell carcinoma of the horn, there were still no reports of the occurrence of the same bilateral in a cow with hormonal disorders.(AU)

Ring depressions in cattle horncores as indicators of traction use - a cautionary note.

In this short communication we call attention to the discovery of a ring depression (a circumferential constriction) in the horncore of a feral bull. This discovery casts doubt on the attribution of similar lesions in archaeological deposits as conclusive evidence of 'cord impressions' resulting from yoking or reining cattle for traction. Drawing upon evidence of horncore depressions in other species, we suggest that they reflect metabolic problems following horn development; additional research is required to test this hypothesis.

Lameness Prevalence and Risk Factors in Large Dairy Farms in Upstate New York. Model Development for the Prediction of Claw Horn Disruption Lesions.

The main objectives of this prospective cohort study were a) to describe lameness prevalence at drying off in large high producing New York State herds based on visual locomotion score (VLS) and identify potential cow and herd level risk factors, and b) to develop a model that will predict the probability of a cow developing claw horn disruption lesions (CHDL) in the subsequent lactation using cow level variables collected at drying off and/or available from farm management software. Data were collected from 23 large commercial dairy farms located in upstate New York. A total of 7,687 dry cows, that were less than 265 days in gestation, were enrolled in the study. Farms were visited between May 2012 and March 2013, and cows were assessed for body condition score (BCS) and VLS. Data on the CHDL events recorded by the farm employees were extracted from the Dairy-Comp 305 database, as well as information regarding the studied cows' health events, milk production, and reproductive records throughout the previous and subsequent lactation period. Univariable analyses and mixed multivariable logistic regression models were used to analyse the data at the cow level. The overall average prevalence of lameness (VLS > 2) at drying off was 14%. Lactation group, previous CHDL, mature equivalent 305-d milk yield (ME305), season, BCS at drying off and sire PTA for strength were all significantly associated with lameness at the drying off (cow-level). Lameness at drying off was associated with CHDL incidence in the subsequent lactation, as well as lactation group, previous CHDL and ME305. These risk factors for CHDL in the subsequent lactation were included in our predictive model and adjusted predicted probabilities for CHDL were calculated for all studied cows. ROC analysis identified an optimum cut-off point for these probabilities and using this cut-off point we could predict CHDL incidence in the subsequent lactation with an overall specificity of 75% and sensitivity of 59%. Using this approach, we would have detected 33% of the studied population as being at risk, eventually identifying 59% of future CHDL cases. Our predictive model could help dairy producers focusing their efforts on CHDL reduction by implementing aggressive preventive measures for high risk cows.

Comprehensive transcriptome profiling of squamous cell carcinoma of horn in Bos indicus.

Squamous cell carcinoma (SCC) of horn is frequently observed in Bos indicus affecting 1% of cattle population and accounting 83.34% of total tumours found. The transcriptome profile of horn cancer (HC) tissue and the matched normal (HN) tissue were analysed by RNA-seq using Roche 454 sequencing. A total of 1 504 900 reads comprising of 612 MB data were used to identify differentially expressed genes using CLC Genomic Workbench. These include up-regulation of KRT6A, KRT6B, KRT6C, KRT14, SFN, KRT84, PI3, COL17A1, ANLN, SERPINB5 and down-regulation of BOLA, SCGB1A1, CXCL17, KRT19, BPIFB1, NR4A1 and TFF3 in HC, which are involved in regulation of gene transcription, cell proliferation, apoptosis, cell survival and metabolic pathways. The qPCR analysis of several targets suggested concordance of gene expression profile with RNA-seq analysis. The present findings would provide basis for further screening of genes and identification of markers for early diagnosis and therapeutic intervention of HC.

Transcriptome analysis and SNP identification in SCC of horn in (Bos indicus) Indian cattle.

Single Nucleotide Polymorphisms (SNPs) have become the marker of choice for genome wide association studies. In order to provide the best genome coverage for the analysis of disease, production and performance traits, a large number of relatively evenly distributed SNPs are needed. The main objective of present work was to identify large numbers of gene-associated SNPs using high-throughput sequencing in squamous cell carcinoma of horn. RNA-seq analysis was conducted on 2 tissues viz. Horn Cancer (HC) and Horn Normal (HN) in Kankrej breed of cattle. A total of 909,362 reads with average read length of 405 bp for HC and 583,491 reads with average read length of 411 bp for HN were obtained. We found 9532 and 7065 SNPs as well as 1771 and 1172 Indels in HC and HN, respectively, from which, 7889 SNPs and 1736 Indels were uniquely present in HC, 5886 SNPs and 1146 Indels were uniquely present in HN and reported first time in Bos indicus, whereas the rest are already reported in Bos taurus dbSNP database. The gene-associated SNPs and Indels were high in upregulated genes of HC as compared to HN. Analysis of differentially expressed genes was identified, these genes are involved in regulation of cell proliferation, apoptosis, gene transcription, cell survival and metabolism through various metabolic pathways. The result of transcriptome expression profiling was validated using Real Time quantitative PCR in nine randomly selected genes. We identified numbers aberrant signaling pathways responsible for carcinogenesis in HC which are also commonly altered in squamous cell carcinoma (SCC) of lung in human being. We conclude that a large number of altered genes and dysfunction of multiple pathways are involved in the development of Horn Cancer. The present findings contribute to theoretical information for further screening of genes and identification of markers for early diagnosis of HC as well as SNPs identified in this report provide a much needed resource for genetic studies in B. indicus and shall contribute to the development of a high density SNP array. Validation and testing of these SNPs using SNP arrays will form the material basis for gene associated SNPs in HC.

Identification of novel exons and transcripts by comprehensive RNA-Seq of horn cancer transcriptome in Bos indicus.

Horn cancer (HC) is a widely prevalent cancer amongst Bos indicus cattle and frequently associated with economic losses due to reduction in draught and milk production capacity. To identify novel transcripts and characterize HC transcriptome, we employed high throughput RNA-Seq analysis of HC and horn normal (HN) tissue. Total of 584,450 and 920,450 high quality reads were analyzed for HN and HC tissue, respectively, resulting in the identification of 16,803 unique genes comprising of 14,754 annotated genes and 2049 novel transcripts. We found 251 significant differentially expressed genes in HC transcriptome, which includes 52 putative novel transcripts. We validated presence of 23 out of 28 novel transcripts by RT-PCR and confirmed differential expression of 12 novel transcripts through RT-qPCR on 4 samples each of HC and HN. Functional analysis revealed that HC is characterized by deregulation of genes involved in epithelial cell differentiation and complement cascade. Our study will serve as a step further in detailed characterization of HC transcriptome and provide firm base to explore and mitigate HC at finer resolution.

A 3.7 Mb deletion encompassing ZEB2 causes a novel polled and multisystemic syndrome in the progeny of a somatic mosaic bull.

Polled and Multisystemic Syndrome (PMS) is a novel developmental disorder occurring in the progeny of a single bull. Its clinical spectrum includes polledness (complete agenesis of horns), facial dysmorphism, growth delay, chronic diarrhea, premature ovarian failure, and variable neurological and cardiac anomalies. PMS is also characterized by a deviation of the sex-ratio, suggesting male lethality during pregnancy. Using Mendelian error mapping and whole-genome sequencing, we identified a 3.7 Mb deletion on the paternal bovine chromosome 2 encompassing ARHGAP15, GTDC1 and ZEB2 genes. We then produced control and affected 90-day old fetuses to characterize this syndrome by histological and expression analyses. Compared to wild type individuals, affected animals showed a decreased expression of the three deleted genes. Based on a comparison with human Mowat-Wilson syndrome, we suggest that deletion of ZEB2, is responsible for most of the effects of the mutation. Finally sperm-FISH, embryo genotyping and analysis of reproduction records confirmed somatic mosaicism in the founder bull and male-specific lethality during the first third of gestation. In conclusion, we identified a novel locus involved in bovid horn ontogenesis and suggest that epithelial-to-mesenchymal transition plays a critical role in horn bud differentiation. We also provide new insights into the pathogenicity of ZEB2 loss of heterozygosity in bovine and humans and describe the first case of male-specific lethality associated with an autosomal locus in a non-murine mammalian species. This result sets PMS as a unique model to study sex-specific gene expression/regulation.

Malignant melanoma of the horn base in a Pygora goat.

A case of malignant melanoma, originating at the base of the left horn, was diagnosed in a white 11-year-old Pygora doe. At necropsy, the goat had an ulcerated, black-pigmented, 2.5-cm mass at the base of the left horn. Firm masses diffusely black on the cut surface were present at the left horn base, subcutaneously over the frontal bones, and infiltrating into the frontal sinuses, the submandibular lymph node, and left parotid salivary gland. The left maxillary premolars and molars were loosened from the periodontia. Multiple black foci of metastasis were observed in the liver. Histologically, the masses consisted predominantly of moderately pleomorphic, polyhedral to spindle cells that contained variable amounts of dark brown intracytoplasmic pigment granules. The tumor cells were positive for Melan A by immunohistochemical staining.

[Ethological, physiological and histological aspects of pain and stress in cattle when being dehorned]. / Ethologische, physiologische und histologische Untersuchungen zur Schmerzbelastung der Rinder bei der Enthornung.

The behaviour of 101 calves was evaluated during (n = 73) and after (n = 28) thermical dehorning without anaesthesia. Cortisol in saliva (n = 68) was measured in 68 calves. A simulation of dehorning was carried out as a control in most of the animals. The area around the horn bud was examined histologically in 20 calves of different ages (newborn until 3-4 months old). Additionally, the influence of dehorning cows with a wire-saw under anaesthesia on behaviour (n = 16), cortisol in saliva (n = 23), and the milk yield was examined. Independent of the calves' age, the horn bud and the surrounding hairy area were well innervated. For all calves dehorning without anaesthesia was a painful experience. During dehorning calves showed distinct pain and defense reactions. Most reactions were observed more often when the calves were dehorned as when dehorning was simulated. The cortisol in saliva was significantly increased after dehorning. In summary, we have to conclude that calves have a well developed nociceptive system from birth on. Therefore calves should only be dehorned using anaesthesia. Despite the anaesthesia, dehorning was stressful for the cows, as measured by a significant increase of cortisol in saliva. Moreover, cows showed pain reactions when the effects of the anaesthesia diminished. Dehorning had only a short effect on the milk yield of the cows.

Experimental poisoning of cattle by the mushroom Ramaria flavo-brunnescens (Clavariaceae): a study of the morphology and pathogenesis of lesions in hooves, tail, horns and tongue.

Seven 2 to 7-mo-old calves were fed the mushroom Ramaria flavo-brunnescens. Clinical signs of toxicosis included salivation, nasal serous discharge, smoothing of the dorsum of the tongue, increased sensitivity in the hooves, reddening of the coronary bands, loss of the long hairs of the tip of the tail, softening of the base of the horns, and progressive weight loss. Affected calves either died or were euthanatized in extremis within 12-37 d from the initiation of feeding. Postmortem examination confirmed changes observed in the live calves. Histopathologic changes were marked in those structures where hard keratinization occurred and there normally is a high uptake of sulfur in the form of cystine during the keratinization process. Toxicosis appeared to alter the metabolism of sulfur-containing amino acids in keratinocytes, particularly cystine, with resultant strength loss in the molecular structure of hard keratin and loosening of the hooves, hairs and horns, and flattening of the lingual filiform papillae.

Cranial asymmetry in a Dall sheep ram (Ovis dalli dalli).

The horns of a 13-yr-old Dall sheep ram (Ovis dalli dalli) from the Sheep Mountain herd in Kluane National Park (Yukon, Canada) had unique characteristics. The right horn was 1,127 mm long, while the left horn was only 861 mm long and compressed and distorted at its base. The reduced growth of the left horn was due to chronic epidermitis and osteomyelitis of the cornual process, which began when the ram was 5 yr of age.

Antler anomalies in tule elk.

Antler anomalies were evident in tule elk (Cervus elaphus nannodes) within 1 yr of reintroduction to Point Reyes, California (USA). These anomalies are consistent with previously described mineral deficiency-induced anomalies in cervids. The elk were judged deficient in copper. Low levels of copper in soils and vegetation at the release site, exacerbated by possible protein deficiency due to poor range conditions, are postulated as likely causes of the antler anomalies.

Further studies on horn aberrations in Dall's sheep (Ovis dalli dalli) from Yukon Territory, Canada.

The prevalence of horn aberrations in Dall's sheep from the Kluane Lake area of Yukon Territory observed during July 1982 represented 1% of the total population and 7% of rams 6 yr or older. Ewes were not considered in these percentages because they were too difficult to inspect by aerial survey. When these data were combined with other data collected from 1977 through 1981, the prevalence equalled 2.4% of the total population and slightly exceeded 16% in mature rams (greater than or equal to 6 yr). The anomaly followed necrosis of the terminal region of the horn core and the sequestering of portions of the core within the sheath as the sheath continued to grow. Sheath that was produced after the core was anatomically altered resulted in abnormal growth patterns of the horn. Rams with aberrant horns could not maintain homeostatic temperatures within horn cores when horns were experimentally exposed to -80 C for 30 min. Histologic examination of superficial and cornual vascular systems did not reveal any structural alterations that would restrict blood flow within cores of affected horns. Examination of museum specimens consisting of 130 skulls from rams and 81 from ewes collected from Alaska, USA, Yukon Territory, Canada, and Northwest Territories, Canada, established only one ewe and no rams with the horn aberration. The skull was from a ewe and had both horns affected and was collected from the Joe River Drainage, Yukon Territory in 1912.

Horn aberrations in Dall's sheep (Ovis dalli) from Yukon Territory, Canada.

Horn aberrations in Dall's sheep from the Kluane Lake area of Yukon Territory were of two basic types. In the more prevalent type, the horn was severed at an annual growth check and the terminal segment of the remaining portion formed a short conical protuberance. The second type had characteristics of the first, but in addition, underwent extreme twisting or torquing during subsequent growth. Sheep with aberrant horns represented 2% of the Kluane Lake population or 14% of harvestable rams (greater than or equal to 6 yr old). Deformed horns, occurring in ewes and rams, were generally observed in animals 6 yr or older. Aberrations followed necrosis of the terminal segment of the horn core: cavitation of the sheath between successive periods of growth or the sequestering of portions of necrotic core within the sheath resulted. All sheep examined (eight) had a normal complement of 54 chromosomes.