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.     

奶牛白细胞黏附缺陷症(BLAD)检测方法的研究进展

奶牛白细胞黏附缺陷症是近年新发现的一种常染色体单基因隐性遗传病,由常染色体上CD18基因单碱基突变(A→G)引起的隐性遗传疾病,隐性基因纯合时导致白细胞表面的β2整合素表达明显减少或缺乏而引起临床发病,患病牛的主要特征是机体免疫力降低、易患病从而影响生产性能,严重影响了奶牛场的经济效益。文章综述了该病检测方法的研究进展。     

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.     

中国荷斯坦牛白细胞粘附缺陷病遗传分析

荷斯坦牛白细胞粘附缺陷病（BLAD）是一种遗传性免疫缺陷病，患病牛出生后，生长发育差，绝大多数在1年内死亡，且不具繁殖和哺育能力。该病的遗传基础为CD18基因编码区383位的A／G点突变导致白细胞表面的β2整合素表达缺陷。目前欧美等奶牛业发达国家都已经建立了完善的BLAD分子检测方法和跟踪监控体系。中国长期从国外进口荷斯坦公牛精液、胚胎或活体，由此可能引进了BLAD基因。本研究采用PCR—RFLP方法对116头可疑中国荷斯坦牛进行了检测．确定了2头BLAD携带者公牛和8头携带者母牛．未发现隐性纯合个体．     

Virus-induced congenital malformations in cattle

Diagnosing the cause of bovine congenital malformations (BCMs) is challenging for bovine veterinary practitioners and laboratory diagnosticians as many known as well as a large number of not-yet reported syndromes exist. Foetal infection with certain viruses, including bovine virus diarrhea virus (BVDV), Schmallenberg virus (SBV), blue tongue virus (BTV), Akabane virus (AKAV), or Aino virus (AV), is associated with a range of congenital malformations. It is tempting for veterinary practitioners to diagnose such infections based only on the morphology of the defective offspring. However, diagnosing a virus as a cause of BCMs usually requires laboratory examination and even in such cases, interpretation of findings may be challenging due to lack of experience regarding genetic defects causing similar lesions, even in cases where virus or congenital antibodies are present. Intrauterine infection of the foetus during the susceptible periods of development, i.e. around gestation days 60–180, by BVDV, SBV, BTV, AKAV and AV may cause malformations in the central nervous system, especially in the brain. Brain lesions typically consist of hydranencephaly, porencephaly, hydrocephalus and cerebellar hypoplasia, which in case of SBV, AKAV and AV infections may be associated by malformation of the axial and appendicular skeleton, e.g. arthrogryposis multiplex congenita. Doming of the calvarium is present in some, but not all, cases. None of these lesions are pathognomonic so diagnosing a viral cause based on gross lesions is uncertain. Several genetic defects share morphology with virus induced congenital malformations, so expert advice should be sought when BCMs are encountered.

Electronic supplementary material

The online version of this article (doi:10.1186/s13028-015-0145-8) contains supplementary material, which is available to authorized users.     

Detection of the nonsense mutation of OPA3 gene in Holstein Friesian cattle with dilated cardiomyopathy in Japan

Bovine dilated cardiomyopathy (DCM) is an autosomal recessive genetic disorder causing congestive heart failure and subsequent death. Recently, a nonsense mutation c.343C>T in the bovine optic atrophy 3 (OPA3) gene had been reported to cause the DCM in Holstein cattle in Switzerland. However, the mutation has not been confirmed in bovine DCM outside Switzerland. Nine Holstein Friesian cows that were macroscopically and histologically diagnosed with or suspected of DCM and 12 control cows kept in Japan were tested for the mutation. The mutation surrounding OPA3 DNA fragment was amplified by PCR and subjected to direct sequences. The homogeneous c.343C>T mutation was proved to occur in all the affected cows and not in the control cows. The present study is the first report of the mutation in the DCM affected cows outside Switzerland.     

Genetic diversity of Anaplasma marginale strains from an outbreak of bovine anaplasmosis in an endemic area

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains. A. marginale genotypes are highly variable in endemic areas worldwide. The genetic composition of A. marginale strains during anaplasmosis outbreaks has been characterized in one study only which reported a single msp1alpha genotype in infected cattle. However, more information is required to characterize whether a single genotype is responsible for an anaplasmosis outbreak or whether multiple genotypes can cause disease in na?ve cattle within a single herd in endemic areas. The aim of this study was to characterize the genetic diversity of A. marginale strains from an outbreak of bovine anaplasmosis in the State of Tamaulipas, Mexico. A. marginale genotypes were characterized at the molecular level using msp4 and msp1alpha gene sequences. The results revealed that several A. marginale genotypes are present in cattle during acute anaplasmosis outbreaks, thus suggesting that mechanical transmission or stochastic biological transmission through equally efficient independent transmission events may explain A. marginale genotype frequency in a cattle herd during acute bovine anaplasmosis outbreaks in endemic areas. The results reported herein corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The development and implementation of anaplasmosis control measures is dependent upon understanding the epidemiology of A. marginale in endemic regions, including the characterization of the genetic diversity of strains that produce outbreaks of bovine anaplasmosis.     

The clinical and pathological findings in two Friesian calves with leukocyte adhesion deficiency

Leukocyte adhesion deficiency is an inherited defect of neutrophil function reported in man, dogs and cattle. In cattle it was first described in 1983 and to date is found only in Holstein Friesians where it is inherited as an autosomal recessive trait. The neu-trophils of affected calves are less able to migrate from blood vessels to sites of inflammation, thus greatly compromising their ability to fight infection.     

瑞士褐牛蜘蛛腿综合征致病位点检测方法的建立

牛蜘蛛腿综合征（Arachnomelia Syndrome,AS）是一种常染色体隐性遗传病,现主要见于德系西门塔尔牛和欧洲瑞士褐牛。患病犊牛先天骨骼系统严重畸形,出生即死亡或出生不久死亡。已证实瑞士褐牛蜘蛛腿综合征是由编码亚硫酸盐氧化酶的基因SUOX外显子4上一个单碱基插入突变c.363-364insG所致。本研究首次建立了瑞士褐牛蜘蛛腿综合征致病基因突变位点分子筛查的方法：荧光引物PCR产物电泳法和PCR产物直接测序法。使用荧光引物PCR产物电泳法对10个品种187头牛样本进行该突变位点的基因型分型,没有发现携带该致病基因的个体。荧光引物PCR产物电泳法判定基因型快速可靠,实验成本较低,适合大规模群体的检测。由于我国曾多次进口瑞士褐牛遗传物质,可能将该致病基因引入本地牛群体。因此对我国相关牛群开展AS隐性不利基因的筛查是必要的,尤其是针对种公牛进行筛查,可避免该致病基因在群体的传播造成重大经济损失。     

Brachygnathia in a herd of Angus cattle

Seven cases of brachygnathia in a single calf crop (28 calves) were observed in an Angus herd. Two of the affected calves, 1 female and 1 male, were necropsied and anatomically described. Breeding histories and results of pedigree analysis involving the 7 affected calves indicated the cause of the defect to be genetic with the mode of inheritance that of a single autosomal recessive gene.     

Nature and cause of bilateral ocular dermoids in Hereford cattle

Nature and cause of bilateral ocular dermoids were investigated by field studies, pedigree analysis, clinical examination, light microscopy, scanning electron microscopy, and transmission electron microscopy. It was determined that ocular dermoids in Hereford cattle are a genetically transmitted defect; characteristics of autosomal recessive and polygenic inheritance were observed. Calves typically were affected bilaterally with multiple, connected ocular growths that clinically, histologically, and ultrastructurally mimicked normal haired skin. Sites most commonly involved included ventro-lateral limbus, third eyelid, medial canthus, eyelid and conjunctiva. Centro-corneal and anterior segmental dermoids also were observed.     

Congenital hydranencephaly and arthrogryposis of Corriedale sheep

SUMMARY: A syndrome of congenital malformations in Corriedale sheep characterised by brachygnathia inferior, campylognathia, tetraemlic arthrogryposis, kyphoscoliosis, hydranencephaly and hypoplasia of the brain stem, cerebellum and spinal cord occurring in various combinations is described. Histologically there was generalised hypomyelinogenesis and hypoplasia of the central nervous system with neurogenic atrophy of skeletal muscle. The syndrome resembled that caused by congenital infection with Akabane virus, however, serological, pathological and epidemiological data indicated that Akabane virus was not involved. The results of a breeding trial suggested that the disorder may be transmitted with an autosomal recessive mode of inheritance.     

Dilated cardiomyopathy (DCM) in dogs--pathological, clinical, diagnosis and genetic aspects

Dilated cardiomyopathy (DCM) is a heart disease which is often found in humans and animals. The age of onset of this progressive disease varies between 3 and 7 years of age. A juvenile form of DCM has been found in Portuguese Water Dogs and Doberman Pinscher Dogs. Some breeds such as Doberman pinscher, Newfoundland, Portuguese Water dog, Boxer, Great Dane, Cocker Spaniel and Irish Wolfhound exhibit a higher prevalence to DCM. There also seems to be a sex predisposition as male dogs are affected more often than female dogs and in Great Danes an X-linked recessive inheritance is likely. In Newfoundland and Boxer an autosomal dominant inheritance was found whereas an autosomal recessive inheritance was described in Portuguese Water Dogs. Atrial fibrillation as a cause or consequence of DCM is assumed for certain breeds. The causes of DCM are widely unknown in dogs. A genetic basis for this heart disease seems to exist. Apart from a few exceptions the mode of inheritance and the possible underlying gene mutations are not known for DCM in dogs. In humans mutations in several genes responsible for DCM have been identified. Comparative genetic analyses in dogs using genes causing DCM in men and a genome-wide scan with anonymus markers were not able to detect causative mutations or genomic regions harboring gene loci linked to DCM. The investigation of the genetic basis of canine DCM may lead to new insights into the pathogenesis of DCM and may result in new therapeutic approaches and breeding strategies.     

Lesions in bovine progressive degenerative myeloencephalopathy ("Weaver") of Brown Swiss cattle

Gross changes and other necropsy findings in 36 purebred Brown Swiss cattle affected with bovine progressive degenerative myeloencephalopathy were nonspecific. Primary microscopic lesions were confined to the central nervous system, specifically the white matter of the spinal cord, axons in some brainstem nuclei, and Purkinje cells of the cerebellar cortex. Spinal cord lesions involved only the white matter and consisted of axonal degeneration, loss of axons and myelin, and status spongiosus. Axonal degeneration was characterized by swelling and fragmentation of the axoplasm or formation of large, discontinuous swellings referred to as spheroids. Lesions were qualitatively similar at all levels, but quantitatively dissimilar in the same funiculi at different levels. Both ascending and descending fibers were involved but correlation to specific fasciculi was not evident. Lesions always were most severe in thoracic spinal cord segments. Little or no astroglial response, no inflammatory response, and no involvement of gray matter were observed in the spinal cord. Cerebellar lesions were limited to selective degeneration and loss of Purkinje cells and occasional swelling of Purkinje cell axons (torpedos) in the granular layer of the cerebellar cortex. Brainstem lesions were inconsistent and limited to occasional axonal swelling in brainstem nuclei. The pathogenesis of bovine progressive degenerative myeloencephalopathy is unknown and possible mechanisms were discussed. The disease exhibits a familial pattern in Brown Swiss cattle and may be hereditary. Extraneural lesions were considered secondary to central nervous system lesions.     

荷斯坦牛脊椎畸形综合征分子诊断方法的建立与应用

脊椎畸形综合征(Complex Vertebral Malformation,CVM)是由常染色体上SLC35A3基因单碱基突变(G→T)引起的隐性遗传疾病,该基因隐性纯合(CV/CV)时奶牛致死,但CVM携带者表现正常,所以CVM携带者公牛可以通过人工授精技术传播CVM缺陷基因。本研究自行设计检测CVM特异性PCR引物,扩增长度为173bp,然后利用PCR-SSCP方法对186个公牛样品和140个母牛样品进行了检测分析,该方法简便快捷、准确率高,使用样品宽泛,适合大样本筛选。研究结果表明,在所检测的样本中,荷斯坦种公牛和母牛CVM携带率分别为11.3%和12.1%,并通过系谱追踪发现,CVM遗传缺陷的共同祖先是美国名牛"Penstate Ivanhoe Star"(USA.1441440,CV)。通过剔除CVM携带者公牛可以有效地控制CVM遗传缺陷,但是,我国许多CVM携带者公牛冻精依然在商业化使用,所以,有效地监控CVM携带者在奶牛群中的状况对CVM防控计划是有益的。     

Canine Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia, affecting both sexes, was present in five of 27 puppies from three father-daughter matings. A purebred Golden Retriever was the common sire. The defect occurred in the left dorsolateral portion of the diaphragm, suggesting failure of closure of the left pleuroperitoneal canal during embryonic development. These findings, and a previous report of a similar defect in neonatal puppies, are consistent with autosomal recessive inheritance of canine diaphragmatic defects.     

Sampling genotype configurations in a large complex pedigree

Many genetic problems can be solved by Monte Carlo method. This often requires sampling genotype configurations over pedigree. Current available samplers are inefficient for large animal pedigrees. A new sampler suitable for large complex pedigrees has been developed and evaluated. The sampler uses simple and iterative peeling algorithms alternately. The sampler was compared to two other samplers on hypothetical pedigree of 79 individuals and recessive disease. The behaviour of the sampler was evaluated in four experimental designs on real bovine pedigree of 907,903 animals. The application of the sampler was also exemplified in identical by descent study.     

A syndrome of hereditary tyrosinemia in mink (Mustela vison Schreb.).

A hereditary disease in mink (Mustela vison Schreb.) leading to death when the affected kits are about six weeks old has been investigated. The disorder is inherited as a simple autosomal recessive character. Strongly elevated plasma tyrosine concentration is an outstanding feature of the disease. An enzyme defect in tyrosine aminotransferase (EC 2.6.1.5) or 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) is considered together with the possibility of a parallel between the disease in mink and the disease tyrosinosis or hereditary tyrosinemia in man.     

Hyperextension of fetlock joints in German Holstein and German Holstein-Limousin crossbred calves

In two different dairy farms six calves exhibiting bilateral flexion of the fetlock joints in front or rear legs were born in 2000 to 2002. Four of the affected calves from the same farm were crossbred between German Holstein cows with red and white coat colour and a bull of the breed Limousin. The other two affected calves born on another farm were purebred German Holsteins with a black and white coat colour. The tests for BVD virus antigen and antibodies were negative in all affected calves. Three of the calves showed a lower selen and a higher glutamate dehydrogenase concentration in the analyses of blood metabolites. Two crossbred calves showed a degeneration of the liver with a progressive periportal fibrosis in a histological examination. In one calf an edema of astrocytes of the central nervous system was seen. The analysis of the pedigrees revealed for the four crossbred calves the Limousin bull as common ancestor and the mothers of the calves as relatives. For the affected purebred German Holstein calves also a sire was identified as a common ancestor. The pedigrees support inheritance through a monogenic autosomal recessive locus or more recessive gene loci with variable expressivity. However, the analysis could not clarify whether different gene loci are responsible for the congenital anomalies observed in the calves from the two farms and thus, the observed anomalies may be different genetic entities. Obvious environmental reasons were not found.     

Spina bifida with associated malformations of the central nervous system in Dorper-cross sheep

Abstract

CASE HISTORY: In 2008, six lambs within a flock of Dorpercross sheep were born with musculoskeletal and neurological disease. Clinical signs included hindlimb weakness, and urinary incontinence.

CLINICAL FINDINGS: All lambs had focal, inverted areas of alopecic skin over the caudal sacrum, and short, often kinked tails. Four affected lambs were subject to euthanasia, and necropsied. On gross examination, the arches of sacral vertebrae were absent, and spinal nerves and meninges were adherent to the overlying subcutis. Other gross lesions included narrow, elongated skulls, herniation of the occipital lobes into the caudal fossas, hydrocephalus, and syringomyelia. One lamb had coning of the cerebellar vermis, but cerebellar herniation through the foramen magnum was not identified.

DIAGNOSIS: Spina bifida, with associated malformations of the central nervous system.

CLINICAL RELEVANCE: Examination of breeding records suggested either an autosomal recessive or partially penetrant autosomal dominant pattern of inheritance. Because of the associated tail lesions it is proposed that the pathogenesis of this syndrome involves a defect in development of the tail bud (secondary neurulation), that tethering of the spinal cord resulted in the clinical signs, and abnormal pressure of the cerebral spinal fluid resulted in the defects in the skull and brain.