Complex vertebral malformation in a holstein calf: report of a case in the USA.

Complex vertebral malformation (CVM), a familial syndrome of Holstein calves, has been reported in aborted fetuses and in prematurely born, stillborn, and neonatal calves. Affected calves have anomalies in the vertebral column, including hemivertebrae, fused and misshapen vertebrae and ribs, scoliosis, and vertebral synostosis. Concurrent low body weight, symmetrical arthrogryposis, and cardiac anomalies have been documented in affected calves. The syndrome was identified and characterized in Holstein cattle in Denmark; however, a global distribution of this genetic disorder is likely based on identification of common ancestral sires widely used for artificial insemination. This is the first documented case of CVM in a Holstein calf in the USA.     

Complex vertebral malformation in holstein calves.

A recently observed lethal congenital defect of purebred Holstein calves is reported. Eighteen genetically related calves were necropsied. One calf had been aborted on gestation day 159, and the others were delivered between day 250 and day 285. Birth weights were reduced. The defect was characterized by shortening of the cervical and thoracic parts of the vertebral column due to multiple hemivertebrae, fused and misshaped vertebrae, and scoliosis. Symmetrical flexures of the carpal joints and the metacarpophalangeal joint in combination with a slight lateral rotation of the phalanges also were present. Similar low-grade arthrogryposis was present in the posterior limbs. Fifty percent of the calves had heart malformation. Other malformations occurred in a few calves. Complex vertebral malformation (CVM) is proposed as the designation for this defect. A genetic etiology is indicated because cases occurred following breeding between genetically related individuals. Two common ancestors were found; both were former elite sires of US Holstein origin. Because of the widespread international use of semen from sires occurring in the pedigrees of affected calves, CVM is expected to occur in several countries.     

Evaluation of the inheritance of the complex vertebral malformation syndrome by breeding studies

To investigate the congenital complex vertebral malformation syndrome (CVM) in Holstein calves, two breeding studies were performed including 262 and 363 cows, respectively. Cows were selected from the Danish Cattle Database based on pedigree and insemination records. Selected cows were progeny of sires with an established heterozygous CVM genotype and pregnant after insemination with semen from another sire with heterozygous CVM genotype. Following calving the breeders should state, if the calf was normal and was requested to submit dead calves for necropsy. In both studies, significantly fewer CVM affected calves than expected were obtained; a finding probably reflecting extensive intrauterine mortality in CVM affected foetuses. The findings illustrate increased intrauterine mortality as a major potential bias in observational studies of inherited disorders.     

Morphological variation of "complex vertebral malformation" in Holstein calves.

A study was performed to investigate the morphological expression of the inherited syndrome "complex vertebral malformation" (CVM) in Holstein calves. A total of 107 late-term aborted, premature, or neonatal calves suspected of having CVM were necropsied and retrospectively analyzed for the causal mutation in the gene SLC35A3. Sixty-two calves were homozygous affected, 16 were heterozygous, and 29 were homozygous normal. Calves affected by CVM were growth retarded. Vertebral lesions identified by radiography were present in 61 cases, of which 58 also had costal malformation. Malformation of the head, primarily in the form of dysplasia or palatoschisis, was present in 15 cases. Bilateral symmetric flexion of the carpal and metacarpophalangeal joints was present in all cases, whereas posterior arthrogryposis was found in 54 cases. Interventricular septal defects occurred in 33 calves, often in combination with other cardiac malformations. A wide spectrum of additional malformations was found. Other congenital syndromes were in most cases distinguishable from CVM on a morphological basis. However, a calf with a prenatal infection with bovine virus diarrhea virus constituted a phenocopy. The study demonstrated that the morphological expression of CVM is wide, but certain aspects, i.e., growth retardation, vertebral malformation, and symmetric arthrogryposis, are almost constant findings. However, cases without vertebral defects and phenocopies constitute a diagnostic problem. A presumptive diagnosis of CVM can in most cases be based on necropsy findings combined with information on descent and paternal CVM genotype, whereas a definitive diagnosis requires genotyping.     

部分中国荷斯坦种公牛脊柱畸形综合征携带状况的检测和分析

摘本研究旨在对部分中国荷斯坦种公牛脊柱畸形综合征(Complex vertebral malformation,CVM)致病基因的携带状况进行筛查.应用错配PCR突变分析技术(PCR mismatch amplification mutation assay,PCR-MAMA)建立了针对CVM致病基因的特异性检测方法.利用PCR-MAMA法检测了154头荷斯坦种公牛,发现了24头CVM阳性个体,阳性率为15.58％.结果显示,应对中国荷斯坦种公牛进行全面的针对CVM的检测.     

Case report: an exceptional case of a monozygotic thoracopagus parasiticus in a German Holstein calf

A black and white German Holstein calf displayed a complex double malformation in shape of a thoracopagus parasiticus. By means of a molecular genetic investigation the genesis of the malformation from one zygote could be demonstrated. Both vertebral columns showed a pronounced lordosis, with the vertebral column of one animal ending in a rudimentary head. Close to this rudiment two derivates of branchial arches were found. The two thoracic cavities merged into one "thorax". In the shared thoracic cavity one heart was found. In its right atrium, a cherry-sized structure was found in which heart- and vascular smooth muscles were demonstrated histologically. The aorta split shortly after its origin to provide both animals with one aorta each. The larger pair of lungs was connected with a trachea leading to the head while the smaller pair of lungs originated from a trachea deriving from the rudimentary head. The diaphragm jejunum and split afterwards. The pedigree of the affected animal showed neither inbreeding nor any other affected animal.     

Identification of complex vertebral malformation carriers in Chinese Holstein.

Complex vertebral malformation (CVM) is a monogenic autosomal recessive hereditary defect of Holstein dairy cattle. It is caused by a point mutation from G to T at the nucleotide position 559 in bovine solute carrier family 35, member 3 gene (SLC35A3), which changes the amino acid sequence of uridine 5'-diphosphate-N-acetylglucosamine transporter protein from a valine to a phenylalanine in position 180. The elite U.S. Holstein sire Penstate Ivanhoe Star was identified as the common ancestor of the current CVM carriers. Because his offspring, mainly those of Carlin-M Ivanhoe Bell, were used in many countries, CVM has potentially spread into China. In the present study, using the polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) technique, 10 CVM carriers were found among 68 at-risk Chinese Holstein bulls, and 282 carriers were found among 602 at-risk cows. The results of this study indicate that the CVM gene exists in the Chinese Holstein population.     

天津地区荷斯坦公牛CVM、DUMPS和CN遗传缺陷检测

本文旨在研究天津地区中国荷斯坦公牛脊椎畸形综合征（Complex vertebral malformation,CVM）、尿苷酸合酶缺乏症（Deftciencv of uridine monophospham synchase,DUMPS）和瓜氨酸血症（Citrullinemia,CN）3种遗传缺陷的携带者比率及系谱来源。通过PIRA—PCR和PCR—RFLP方法分别对天津奶牛发展中心参加全国青年公牛联合后裔测定和国家良种补贴项目的110头荷斯坦公牛进行了CVM、DUMPS和CN三种遗传缺陷检测。共发现6头CVM隐性有害基因携带公牛,携带者比例为5．45％,隐性有害等位基因频率为2．72％。经过系谱分析,其中4头CVM携带者均为美国公牛Carlin—MIvanhoeBell的后代,另外2头因系谱不完整而无法查询。未检测到DuMPs和CN隐性有害基因携带者。基于此,我国有必要尽快建立荷斯坦牛隐性遗传缺陷监控体系并进行系谱标注,避免携带公牛进入后裔测定和良种补贴项目,以逐步降低我国奶牛群体中遗传缺陷隐性等位基因频率。     

A novel method for rapid and reliable detection of complex vertebral malformation and bovine leukocyte adhesion deficiency in Holstein cattle

Background: Complex vertebral malformation (CVM) and bovine leukocyte adhesion deficiency (BLAD) are two autosomal recessive lethal genetic defects frequently occurring in Holstein cattle, identifiable by single nucleotide polymorphisms. The objective of this study is to develop a rapid and reliable genotyping assay to screen the active Holstein sires and determine the carrier frequency of CVM and BLAD in Chinese dairy cattle population. Results: We developed real-time PCR-based assays for discrimination of wild-type and defective alleles, so that carriers can be detected. Only one step was required after the DNA extraction from the sample and time consumption was about 2 hours. A total of 587 Chinese Holstein bulls were assayed, and fifty-six CVM-carriers and eight BLAD-carriers were identified, corresponding to heterozygote carrier frequencies of 9.54% and 1.36%, respectively. The pedigree analysis showed that most of the carriers could be traced back to the common ancestry, Osborndale Ivanhoe for BLAD and Pennstate Ivanhoe Star for CVM. Conclusions: These results demonstrate that real-time PCR is a simple, rapid and reliable assay for BLAD and CVM defective allele detection. The high frequency of the CVM allele suggests that implementing a routine testing system is necessary to gradually eradicate the deleterious gene from the Chinese Holstein population.     

Occipito-atlanto-axial malformation with atlanto-axial subluxation in an ataxic calf

Occipito-atlanto-axial malformation with atlanto-axial subluxation was diagnosed radiographically in a 4-day-old Holstein calf with a short neck, symmetric ataxia, and tetraparesis, indicative of a cervical spinal cord lesion. Necropsy confirmed the clinical diagnosis and revealed ribs on C-7, with partial fusion of C-7 and T-1. Histologic examination revealed focal degeneration of the spinal cord in the second cervical segment at the level of the atlanto-axial joint and extensive secondary neuronal fiber degeneration possibly caused by spinal cord trauma that occurred in utero.     

Evaluation of Cervical Spinal Fusion as a Treatment in the Equine "Wobbler" Syndrome

Cervical vertebral interbody fusion was performed on 30 horses affected with cervical vertebral malformation (CVM) or "wobbles" to assess the efficacy of the surgery in either preventing progression of or reversing the neurologic deficit induced by the syndrome. Evaluation of each horse prior to surgery included clinical, radiographic, neurologic, and laboratory examination. Subjects ranged from 3 months to 8 years of age, and included 22 males and 8 females of various breeds. Three normal horses were included as controls. The Cloward method of cervical fusion was used to achieve stabilization at the affected vertebral articulation. Horses were reexamined 3 months after surgery. Clinical improvement was seen in 90% (27 of 30) of the cases. Four horses were returned to training, which had been interrupted by onset of symptoms. Seven were returned to owners as breeding animals. Twelve horses were kept for long-term studies. Seven horses were sacrificed to examine the surgical site. In 6 of the 7 horses on which necropsy was performed, the most severe histologic lesion in the spinal cord could be predicted from lesions seen radiographically. The spinal cords of control animals were normal. Osseous fusion was dependent on the completeness of removal of disc fibrocartilage during surgery. We conclude that clinical improvement in some horses affected with CVM can be achieved by cervical fusion.     

A novel method for rapid and reliable detection of complex vertebral malformation and bovine leukocyte adhesion deficiency in Holstein cattle

Background

Complex vertebral malformation (CVM) and bovine leukocyte adhesion deficiency (BLAD) are two autosomal recessive lethal genetic defects frequently occurring in Holstein cattle, identifiable by single nucleotide polymorphisms. The objective of this study is to develop a rapid and reliable genotyping assay to screen the active Holstein sires and determine the carrier frequency of CVM and BLAD in Chinese dairy cattle population.

Results

We developed real-time PCR-based assays for discrimination of wild-type and defective alleles, so that carriers can be detected. Only one step was required after the DNA extraction from the sample and time consumption was about 2 hours. A total of 587 Chinese Holstein bulls were assayed, and fifty-six CVM-carriers and eight BLAD-carriers were identified, corresponding to heterozygote carrier frequencies of 9.54% and 1.36%, respectively. The pedigree analysis showed that most of the carriers could be traced back to the common ancestry, Osborndale Ivanhoe for BLAD and Pennstate Ivanhoe Star for CVM.

Conclusions

These results demonstrate that real-time PCR is a simple, rapid and reliable assay for BLAD and CVM defective allele detection. The high frequency of the CVM allele suggests that implementing a routine testing system is necessary to gradually eradicate the deleterious gene from the Chinese Holstein population.     

Effects of complex vertebral malformation on fertility in Swedish holstein cattle

Complex vertebral malformation (CVM) is an autosomal recessive inherited defect in the Holstein breed. It causes intra-uterine mortality through the entire gestation period leading to repeat breeding and involuntary culling of cows and thereby economic losses. The defect was first reported in Denmark in 1999 and a direct DNA test for the defect has been available since February 2001. The aim of this study was to investigate if Holstein bulls heterozygous for the CVM gene had reduced reproductive performance, measured as non-return rate (NRR) and in a daughter fertility index. All genotyped Swedish Holstein bulls born between 1995 and 1999 were included. Altogether 228 bulls were analysed, of which 53 bulls, i.e. 23%, were confirmed CVM carriers. A statistically significant difference between carriers and non-carriers in the relative breeding value for NRR was observed for 168 days NRR (101.1+/-0.9 vs. 103.1+/-0.6, p<0.05). There was no difference for 28 days NRR whereas the difference approached significance for 56 days NRR. No significant effect of the paternal CVM genotype on the daughter fertility index was shown probably due to the complexity of traits this index is composed of. In conclusion, the study showed that carriers of the CVM defect have an inferior NRR compared with non-carriers.     

A method for detecting complex vertebral malformation in Holstein calves using polymerase chain reaction-primer introduced restriction analysis.

Complex vertebral malformation (CVM), a hereditary lethal disease in Holstein calves, is characterized by complex anomalies of the vertebral column and limbs in an aborted fetus and in prematurely born, stillborn, and neonatal calves. The mode of inheritance of CVM is autosomal recessive, and CVM is caused by a point mutation from G to T at nucleotide position 559 of the bovine solute carrier family 35 member 3 (SLC35A3) gene. Although an allele-specific polymerase chain reaction (AS-PCR) is a useful method for diagnosis of CVM, the AS-PCR requires selected DNA polymerases and strictly controlled reaction conditions to obtain reliable results. Therefore, an alternative screening method for the CVM gene would be useful. Polymerase chain reaction-primer introduced restriction analysis (PCR-PIRA) is a method that can be used for detecting a single nucleotide mutation in any gene without a restriction site around the mutation site. In this study, primers were designed to introduce PstI or EcoT22 sites into PCR products from the wild-type and CVM alleles, respectively. The wild-type allele, a heterozygote, and a homozygote of the CVM allele could be discriminated by restriction fragment length polymorphism analysis. Specific introduction of restriction sites into PCR products depending on the change in a single nucleotide of template was shown using a variety of DNA polymerases and PCR machines. Therefore, the PCR-PIRA technique using primers designed in this study might provide a more useful method for extensive screening of CVM.     

Genetic Description of Factor XI Deficiency in Holstein Semen in Western Japan

Factor XI deficiency was detected in Holstein cows and mummified foetuses in Japan; however, no report is available about the occurrence of Factor XI deficiency in Holstein semen in Japan. Five hundred cows in twelve dairy farms in Hiroshima Prefecture, Japan were under the study. Genomic DNA was extracted from the cows using a commercial DNA kits and screened to Factor XI mutation. Based on the information of the carrier cows found in the cattle population, four Holstein bulls were analysed for Factor XI mutation. DNA was extracted from bull's semen using phenol chloroform method. Extracted genomic DNA of the bull's semen was typed for Factor XI using specific polymerase chain reaction (PCR) primers. The resultant PCR was sequenced using big dye terminator sequencing method. The pedigree of the bulls was investigated. Furthermore, the inheritance of Factor XI mutation to next generation was estimated. Out of the 500 cows, five were heterozygous to Factor XI. Moreover, out of the four bulls, one was found to carry the mutation of Factor XI; it was also a complex vertebral malformation (CVM) carrier. In DNA sequencing, the insertion mutation of 76 bp of poly-adenine that characterizes the Factor XI deficiency was detected in the carrier bull as well as the carrier cows. Pedigree analysis of the carrier bull revealed that his father and mother ID were 2247419A and 14189172A, respectively, that originated from USA Holstein. Out of six daughter cows born to the carrier bull, one cow (16.6%) inherited Factor XI mutation, while three of them (50.0%) inherited CVM mutation. Autosomal recessive genes that affect cow's reproduction have a particular concern to dairy industry. To our knowledge this is the first report of Factor XI mutation in Holstein semen in Japan.     

Brachyspina syndrome in a Holstein calf.

A stillborn premature Holstein calf was necropsied. The calf had severely reduced body weight and shortening of the vertebral column. The limbs appeared long and thin. Radiography and computer tomography scanning were performed, demonstrating malformation of the vertebral column. In addition, malformation of the kidneys, testicles, and heart was found at necropsy. Histologically, absence or incomplete development of intervertebral discs and complete or partial fusion of the epiphyses of adjacent vertebrae were found. In some areas, diaphyses of adjacent vertebrae were fused due to absence of epiphyses and intervertebral discs. Disturbed ossification of epiphyses in the appendicular skeleton was also present. The etiology of this syndrome was not identified. An inherited cause is possible due to a close relationship between the parents.     

Hypervitaminosis A in a cat.

A 10-year-old cat that was kept on a diet consisting largely of raw liver was evaluated because of lethargy, partial anorexia, and weight loss of several months' duration. The cat's head and neck were rigidly extended, and a hard mass was palpable in the ventral cervical region. Cervical and thoracic radiography revealed proliferative bony lesions of the cervical and thoracic vertebrae as well as of the sternum and costal cartilages. Serum vitamin A concentration was 4 times normal. For reasons unrelated to hypervitaminosis A, euthanasia and necropsy were performed 6 months after evaluation. The skull and the cervical and first few thoracic vertebrae were rigidly fused, and the vertebral architecture was altered by deposition of new bone. The sternum and costal cartilages were similarly affected. The historical, physical, radiographic, laboratory, and postmortem findings were consistent with the diagnosis of hypervitaminosis A. On the basis of findings in this cat, hypervitaminosis A should be suspected in any sick cat fed a diet consisting partly or completely of raw liver.     

Ovarian Cyclicity and Reproductive Performance of Holstein Cows Carrying the Mutation of Complex Vertebral Malformation in Japan

This study was carried out on 71 lactating Holstein Friesian cows to investigate the resumption of ovarian cyclicity postpartum and the reproductive performance in cows carrying the mutation of complex vertebral malformation (CVM) compared with control ones. The cows were distributed in two dairy farms in Hiroshima Prefecture, Western Japan. Blood samples were collected from the cows to detect carrier cows with CVM mutation. Furthermore, plasma samples were collected weekly after calving from control cows (n = 10) and CVM carrier cows (n = 10), until 10 weeks postpartum to investigate the day of first ovulation and the resumption of ovarian cyclicity postpartum. The reproductive parameters were investigated and compared with control and CVM carrier cows. Thirty‐six cows were diagnosed to be CVM carriers by DNA examination and confirmed later by DNA sequencing. The pedigree analysis of the carrier cows revealed that they were daughters of six types of CVM carrier semen that still was used in dairy farms in Western Japan. In terms of reproductive indices, there were no significant differences between the control and the CVM carrier cows on the day of the first ovulation postpartum and the interval from calving to first insemination. However, CVM carrier cows significantly required more inseminations per conception and showed a significantly longer period to conception and subsequent calving than control ones. In conclusion, the reproductive performance of the CVM carrier cows was lowered through conception failure that might indicate the occurrence of intra‐uterine mortality in those cows.     

Screening for bovine leukocyte adhesion deficiency,deficiency of uridine monophosphate synthase,complex vertebral malformation,bovine citrullinaemia,and factor XI deficiency in Holstein cows reared in Turkey

Background

Bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins.

Methods

Genomic DNA was obtained from blood and the amplicons of BLAD, DUMPS, CVM, BC and FXID were obtained by using PCR. PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. These digested products and PCR product of FXID were analyzed by agarose gel electrophoresis stained with ethidium bromide. CVM genotypes were detected by DNA sequencing. Additionally, all genotypes were confirmed by DNA sequencing to determine whether there was a mutant allele or not.

Results

Fourteen BLAD, twelve CVM and four FXID carriers were found among the 350 Holstein cows examined, while carriers of DUMPS and BC were not detected. The mutant allele frequencies were calculated as 0.02, 0.017, and 0.006 for BLAD, CVM and FXID, respectively with corresponding carrier prevalence of 4.0% (BLAD), 3.4% (CVM) and 1.2% (FXID).

Conclusion

This study demonstrates that carriers of BLAD, CVM and FXID are present in the Turkish Holstein population, although at a low frequency. The actual number of clinical cases is unknown, but sporadic cases may appear. As artificial insemination is widely used in dairy cattle breeding, carriers of BLAD, CVM and FXID are likely present within the population of breeding sires. It is recommended to screen breeding sires for these defective genes in order to avoid an unwanted spread within the population.     

Unusual case of spina bifida in a Friesian cross calf

A case of spina bifida in a Friesian cross calf is described. The case was unusual as the calf was alive and ambulatory. There was no locomotor disturbance but a small skin defect was present in the thoracic region with anomalies of the thoracic vertebral laminae 9 and 10 and the associated ribs. Radiographic studies and post mortem findings are reported to indicate the extent of the lesion.