Multiple independent de novo mutations are associated with the development of schistosoma reflexum, a lethal syndrome in cattle.

Schistosoma reflexum (SR) is a lethal congenital syndrome characterized by U-shaped dorsal retroflexion of the spine and exposure of abdominal viscera. SR is usually associated with severe dystocia. The syndrome is thought to be inherited as a Mendelian trait. We collected a series of 23 SR-affected calves from four breeds (20 Holstein, one Red Danish, one Limousin, one Romagnola) and performed whole-genome sequencing (WGS). WGS was performed on 51 cattle, including 14 cases with parents (trio-based; Group 1) and nine single cases (solo-based; Group 2). Sequencing-based genome-wide association studies with 20 Holstein cases and 154 controls showed no association (above Bonferroni threshold; P-value<3 ×10-09). Assuming a monogenic recessive inheritance, no region of shared homozygosity was observed, suggesting heterogeneity. Alternatively, the presence of possible dominant acting de novo mutations were assessed. In Group 1, heterozygous private variants, absent in both parents, were found in seven cases. These involved the ACTL6A, FLNA, GLG1, IQSEC2, MAST3, MBTPS2, and MLLT1 genes. In addition, heterozygous private variants affecting the genes DYNC1LI1, PPP2R2B, SCAF8, SUGP1, and UBP1 were identified in five cases from Group 2. The detected frameshift and missense variants are predicted to cause haploinsufficiency. Each of these 12 affected genes belong to the class of haploinsufficient loss-of-function genes or are involved in embryonic and pre-weaning lethality or are known to be associated with severe malformation syndromes in humans and/or mice. This study presents for the first time a detailed genomic evaluation of bovine SR, suggesting that independent de novo mutations may explain the sporadic occurrence of SR in cattle.

Identification of small and large genomic candidate variants in bovine pulmonary hypoplasia and anasarca syndrome.

The pulmonary hypoplasia and anasarca syndrome (PHA) is a congenital lethal disorder, which until now has been reported in cattle and sheep. PHA is characterized by extensive subcutaneous fetal edema combined with hypoplasia or aplasia of the lungs and dysplasia of the lymphatic system. PHA is assumed to be of genetic etiology. This study presents the occurrence of PHA in two different cattle breeds and their genetic causation. Two PHA cases from one sire were observed in Slovenian Cika cattle. Under the assumption of monogenic inheritance, genome-wide homozygosity mapping scaled down the critical regions to 3% of the bovine genome including a 43.6 Mb-sized segment on chromosome 6. Whole-genome sequencing of one case, variant filtering against controls and genotyping of a larger cohort of Cika cattle led to the detection of a likely pathogenic protein-changing variant perfectly associated with the disease: a missense variant on chromosome 6 in ADAMTS3 (NM_001192797.1: c.1222C>T), which affects an evolutionary conserved residue (NP_001179726.1: p.(His408Tyr)). A single PHA case was found in Danish Holstein cattle and was whole-genome sequenced along with its parents. However, as there was no plausible private protein-changing variant, mining for structural variation revealed a likely pathogenic trisomy of the entire chromosome 20. The identified ADAMTS3 associated missense variant and the trisomy 20 are two different genetic causes, which shows a compelling genetic heterogeneity for bovine PHA.

Congenital Cutaneous Panadnexal Papillomatous Hamartomas in a Calf.

Several cauliflower-like alopecic masses arose on the head of an otherwise healthy, full-term newborn Jersey × Belgian blue heifer, which was humanely destroyed shortly after birth due to the severity of the skin lesions. Microscopically, the masses were composed of multiple papillary projections displaying well-differentiated sebaceous glands surrounded by a moderate number of well-developed sweat glands, as well as embryonic and fully developed, but dysplastic hair follicles. Thick branching connective tissue stalks supported these adnexal components. The papillated surface, the predominance of sebaceous glands, the presence of embryonic hair follicles and the well-differentiated sweat glands were compatible with nevus sebaceous, a rare form of cutaneous hamartoma described in man, dogs, cats and cattle. However, the cauliflower-like growth pattern, the presence of supportive thick branching connective tissue stalks and the relative abundance of dysplastic hair follicles in association with nevus sebaceous has not been described in the human or veterinary literature. A diagnosis of panadnexal papillomatous hamartoma was made in this case.

Bacterial invasion of the uterus and oviducts in bovine pyometra.

Pyometra is a common disease of cattle that causes infertility and thereby financial losses to the cattle industry. Bacteria involved in the development and progression of pyometra have been investigated by microbial culture but their tissue invading abilities, which is an important aspect of bacterial pathogenicity and development of lesions, have not been investigated. Bacterial invasion of the uterus and oviducts was studied in 21 cows diagnosed with pyometra at the time of slaughter by applying fluorescence in situ hybridization using probes targeting 16S ribosomal RNA of Fusobacterium necrophorum, Porphyromonas levii, Trueperella pyogenes and the overall bacterial domain Bacteria. Fusobacterium necrophorum and P. levii were found to invade the endometrium, especially if the endometrium was ulcerated, and penetrated deep into the lamina propria. These species co-localized within the tissue thus indicating a synergism. Trueperella pyogenes did not invade the uterine tissue. In addition to endometrial lesions, most cows with pyometra also had salpingitis but without significant bacterial invasion of the oviductal wall.

Bilateral oblique facial clefts, rudimentary eyes and hydrocephalus in an aborted equine foetus.

Knowledge of congenital malformations and their causes in horses is generally sparse. Such conditions require more scientific attention to improve their diagnostics and inform prevention strategies. Here, a unique syndrome of bilateral oblique facial clefts (meloschisis), rudimentary eyes and hydrocephalus is reported in an equine foetus spontaneously aborted at gestation day 224. The cause of abortion was considered to be intrauterine death caused by umbilical cord torsions and subsequent compromised blood flow, but the aetiology of the malformation could not be determined. A detailed history, which includes exposure to a range of pharmaceutical compounds during the early stages of pregnancy, is provided and emphasizes the need for accurate recording of treatments in pregnant animals.

Presence and localization of bacteria in the bovine endometrium postpartum using fluorescence in situ hybridization.

The aim of this study was to investigate bacterial invasiveness of the bovine endometrium during the postpartum period. Fluorescence in situ hybridization was applied to endometrial biopsies using probes for Fusobacterium necrophorum, Porphyromonas levii, Trueperella pyogenes, Escherichia coli and a probe for bacteria in general (the overall domain Bacteria) to determine their tissue localization. Holstein cows were sampled at three time points postpartum (T1: 4-12 days postpartum, T2: 24-32 days postpartum and T3: 46-54 days postpartum). At T1, cows were clinically scored as having a uterine infection based on presence of a brownish, fetid vaginal discharge or as normal if having normal lochia. An endometrial biopsy was taken from all cows at T1 (n = 57). Endometrial biopsies were taken from the same cows at T2 and T3 if allowed by the size of the cervical canal and if the cow had not been inseminated. Fifty and 39 biopsies were obtained at T2 and T3, respectively. The biopsies were evaluated for inflammation and for presence and localization of bacteria. When analyzed by the probe for the entire domain Bacteria, bacteria were found in most biopsies irrespectively of time (T1: 79.0%, T2: 82.0%, T3: 89.7%). Fusobacterium necrophorum and Porphyromonas levii were often present in the endometrium at T1 (61.1% and 47.8%, respectively), but the prevalence decreased significantly over time. Trueperella pyogenes and Escherichia coli were less prevalent at T1 (8.8% and 10.5%, respectively) and their prevalence also decreased significantly over time. Fusobacterium necrophorum and Porphyromonas levii were often co-localized intraepithelially or in the lamina propria. Trueperella pyogenes and Escherichia coli were located only on the endometrial surface. Due to the high prevalence of tissue invasiveness, these findings emphasize the importance of Fusobacterium necrophorum and Porphyromonas levii in postpartum uterine disease of cattle and indicate that tissue invasiveness is an important aspect of the pathogenesis.

Sarcocystosis in a stillborn lamb.

Confirmed congenital sarcocystosis has been reported extremely rarely in domestic ruminants. Sarcocystosis was diagnosed in a stillborn lamb with microscopic lesions predominantly in the central nervous system and placenta. Encephalitis was characterized by multiple foci of glial nodules some with central necrosis, perivascular cuffing and vascular occlusion, while placental lesions consisted of multifocal necroses, inflammation and mild calcification. Immature and mature schizonts were found in vascular endothelium of several organs. It is suggested that the protozoa were Sarcocystis tenella based on their morphology, location and as this is the most pathogenic Sarcocystis sp. parasitizing sheep.

Intrauterine idiopathic amputation of the head of a porcine foetus.

An anencephalic full-term porcine foetus accompanied by a mummified head was submitted for examination. The neck almost entirely lacked skin and was covered by granulation tissue as were the exposed parts of the spine and spinal cord. The case represents a rare case of intrauterine amputation. A definitive cause could not be established because the placenta was not available. The most likely cause is strangulation of the neck. Such strangulation could be due to a defect of the allantoamnion with herniation of the foetal head or entanglement by amniotic constriction bands.

A molecular study of congenital erythropoietic porphyria in cattle.

Previous studies have shown that congenital erythropoietic porphyria (CEP) in cattle is caused by an inherited deficiency of the enzyme uroporphyrinogen III synthase (UROS) encoded by the UROS gene. In this study, we have established the pedigree of an extended Holstein family in which the disease is segregating in a manner consistent with autosomal recessive inheritance. Biochemical analyses demonstrated accumulation of uroporphyrin, thus confirming that it is indeed insufficient activity of UROS which is the cause of the disease. We have therefore sequenced all nine exons of UROS in affected and non-affected individuals without detecting any potential causative mutations. However, a single nucleotide polymorphism (SNP) located within the spliceosome attachment region in intron 8 of UROS is shown to segregate with the disease allele. Our study supports the hypothesis that CEP in cattle is caused by a mutation affecting UROS; however, additional functional studies are needed to identify the causative mutation.

Exonization of a LINE1 fragment implicated in X-linked hypohidrotic ectodermal dysplasia in cattle.

A case of X-linked hypohidrotic ectodermal dysplasia (XHED) was identified in a family of Danish Red Holstein cattle. The ectodysplasin-signalling protein (EDA) is known to be central in the normal development of ectodermal structures, and mutations in the ectodysplasin A (EDA) gene have been reported to cause XHED. In this study, we analysed different EDA transcript variants in affected and unaffected cattle and identified a new transcript variant including a LINE1-derived pseudoexon between EDA exons 1 and 2. The 161-bp-long pseudoexon introduces a shift in reading frame and a premature stop codon early in EDA exon 2 and is probably the cause of XHED in this Danish Red Holstein family.

Dynamics of relationship between the presence of Coxiella burnetii DNA, antibodies, and intrinsic variables in cow milk and bulk tank milk from Danish dairy cattle.

Milk samples of 12 Danish dairy herds were collected 3 times during an 11-mo period and tested for Coxiella burnetii DNA by real-time PCR, detecting the IS1111 element, and for the presence of antibodies against the bacterium by ELISA. On average, 25% of 1,514 samples were seropositive and 32% were positive for C. burnetii DNA. Among the 485 DNA-positive samples, quantification cycle values ranging from 15.8 to 37.8 were found. Test sensitivity did not increase after DNA extraction from the cream fraction compared with full milk. The relationship between antibody levels and bacterial shedding was investigated among 166 cows from 9 herds. The prevalence levels of C. burnetii DNA and antibodies in the herds were found to be rather stable for 6 of the herds. The test results were highly influenced by results obtained 3 to 7 mo earlier. A significant association between the antibody titer and the DNA shedding level at the same and the preceding visit was found. In addition, a significant association between the antibody titer and the antibody titers 3 to 11 mo earlier was found. A multivariable analysis identified a significant increase in C. burnetii DNA shedding with increasing parity and increasing protein concentration in milk. The antibody levels in bulk tank milk and prevalence levels of C. burnetii DNA and antibodies in individual cow milk samples were correlated. A significant correlation was also found between the quantification cycle values of the cow samples (weighted according to milk yield) and the C. burnetii concentration in bulk tank milk.

Maternal endometrial oedema may increase perinatal mortality of cloned and transgenic piglets.

The perinatal mortality of cloned animals is a well-known problem. In the present retrospective study, we report on mortality of cloned transgenic or non-transgenic piglets produced as part of several investigations. Large White (LW) sows (n = 105) received hand-made cloned LW or minipig blastocysts and delivered either spontaneously or after prostaglandin induction followed by either Caesarean section or vaginal birth. The overall pregnancy rate was 62%, with 26% of pregnancies terminating before term. This resulted in 48 deliveries. The terminated pregnancies consisted of 12 abortions that occurred at 35 ± 2 days gestation and five sows that went to term without returning to heat and then by surgery showed the uterus without fetal content. The gestation length was for sows with LW piglets that delivered by Caesarean section or vaginally was 115.7 ± 0.3 and 117.6 ± 0.4 days, respectively. In sows with minipiglets, the gestation length for those delivered by Caesarean section or vaginally 114.4 ± 0.2 and 115.5 ± 0.3 days, respectively. Of the 34 sows that delivered vaginally, 28 gave birth after induction, whereas 6 farrowed spontaneously. Of the 14 sows that delivered after Caesarean section and in the five empty sows, the endometrium and placenta showed severe oedema. Piglet mortality following vaginal delivery was higher than after Caesarean section (31% v. 10%, respectively; P < 0.001). When vaginal delivery occurred spontaneously, the stillborn rate was greater than after induced delivery (56% v. 24%, respectively; P < 0.0001). Internal organ weights were recorded for seven cloned LW piglets and six normal piglets. The relative weight of the heart, liver, kidneys and small intestine was found to be reduced in the cloned piglets (P < 0.05). The present study demonstrates extensive endometrial oedema in sows pregnant with cloned and transgenic piglets, as well as in empty recipients, at term. The growth of certain organs in some of the cloned piglets was reduced and the rate of stillborn piglets was greater in cloned and transgenic piglets delivered vaginally, possibly because of oedema of the fetal-maternal interface.

Endocarditis-associated brain lesions in slaughter pigs.

Left-sided valvular endocarditis (LSVE) is a common finding in slaughter pigs. The lesion is often associated with renal thromboembolism, but information on embolization to other organs is sparse. This study focuses on the presence and type of endocarditis-associated brain lesions (EABLs). The brains of 20 slaughter pigs with spontaneously arising LSVE and 11 controls were examined by sectioning half of a formalin-fixed brain into 4mm slices for histological examination. The aetiology of the endocarditis was determined by bacteriological and, in some cases, by fluorescence in-situ hybridization examinations. These examinations identified 11 cases of Streptococcus suis, six cases of Erysipelothrix rhusiopathiae, one Streptococcus spp. and two cases that remained aetiologically undetermined. One of the S. suis cases had a dual infection with S. suis in the aortic valve lesions and Streptococcus dysgalactiae subsp. equisimilis in the atrioventricular valve lesions. Renal infarcts were present in eight cases. Focal encephalitis was found in 12 cases, with the number of lesions ranging from one to 11. Most pigs had less than four microscopical lesions. Acute lesions were characterized by focal microabscesses without observable bacteria. Chronic lesions were characterized by astrocytosis and focal accumulation of mononuclear leucocytes. An infarct was observed in one animal. Perivascular inflammation was seen in 14 cases, mostly as two or three lesions, while focal leptomeningitis was found in eight cases. EABLs are therefore common in slaughter pigs with LSVE. The number of lesions per animal is small, which may explain the limited attention paid to this sequela of LSVE. EABLs have rarely been reported in domestic animals and mostly in patients with neurological signs. The frequent occurrence of EABLs in slaughter pigs suggests that this pathology should be investigated in other animal species with LSVE.

Digital necrobacillosis in Norwegian wild tundra reindeer (Rangifer tarandus tarandus).

Outbreaks of digital necrobacillosis in Norwegian wild tundra reindeer (Rangifer tarandus tarandus) are described. The outbreaks occurred in late summer and autumn 2007 and 2008, subsequent to periods with an unusually high number of days with precipitation and high air temperature. Lesions were generally restricted to one foot and the disease incidence was highest in calves. Single limbs from 20 animals and six whole carcasses were submitted for laboratory examination. Gross lesions were characterized by swelling of the fetlock to coronary band area and cutaneous sinus tracts with sparse discharge of pus. Subcutaneous tissue was inflamed and oedematous with focal necrosis. Tendons, tendon sheaths, joints and periosteum of the digital bones were often affected. Animals shot during winter showed severe chronic periostitis and osteomyelitis and necrotizing deforming arthritis. Microscopically, skin lesions were characterized by deep ulcers with centrally located necrotic tissue, bordered by a zone of oedema and intense inflammation with granulation tissue and fibrosis. Necrosis, suppurative inflammation and oedema were found in the synovial membranes, tendons and tendon sheaths. Digital bone lesions were characterized by necrosis, fibrosis and extensive bone proliferation. Vasculitis and thrombosis were common in all lesions. Elongate filamentous gram-negative bacteria in necrotic lesions from all animals were identified as Fusobacterium necrophorum by fluorescence in-situ hybridization. F. necrophorum was cultured from the foot lesions of six animals. Five of these isolates were examined by 16S rRNA sequencing. The sequences were identical and differed from all other strains listed in GenBank. These results are consistent with circulation of a reindeer-adapted pathogenic strain of F. necrophorum in the wild reindeer population, causing outbreaks of digital necrobacillosis following warm and humid summers.

Early pathogenesis in porcine proliferative enteropathy caused by Lawsonia intracellularis.

The intestinal bacterium Lawsonia intracellularis, the cause of proliferative enteropathy (PE) in pigs, is believed to infect mitotically active epithelial cells of the intestinal crypts and then multiply and spread in these cells as they divide. Further spread of infection is thought to occur by shedding of bacteria from infected crypts followed by infection of new crypts. The early stages of the pathogenesis of PE, from 0 to 48 hours post-infection (hpi), have not been studied in vivo. In the present study pigs were inoculated with L. intracellularis and killed from 12 hpi to 5 days post-infection (dpi). The localization of L. intracellularis was determined immunohistochemically and by fluorescence in-situ hybridization. At 12 hpi L. intracellularis was found within epithelial cells at the tips of villi, indicating infection of a range of epithelial cells including mature differentiated enterocytes. Furthermore, early invasion of the intestinal connective tissue was observed; with the presence of single bacteria in the lamina propria 12 hpi, and with a further spread of bacteria in the lamina propria observed at 5 dpi, suggesting an active role for the lamina propria in the course of infection.

Ovine fetal necrobacillosis.

The association of Fusobacterium necrophorum with late term abortion in sheep is reported. The bacterium was not culturable, but was identified in five cases by fluorescence in-situ hybridization (FISH) with an oligonucleotide probe specifically targeting 16S rRNA in F. necrophorum. Gross lesions were found in several tissues. Histologically, placental lesions were characterized by locally diffuse infiltration of neutrophils, closely associated with abundant small Gram-negative and FISH-positive rods, thrombosis and necrosis. Lesions in the fetal-maternal interface were multifocal and consisted of villous necrosis and suppurative inflammation. Spread to the fetus from the placenta appeared to occur in two ways. Some fetuses had multifocal necrotizing hepatitis consistent with haematogenous spread through the umbilical vein; further dissemination to other organs occurred. Transplacental spread and infection of the fetus through the amniotic fluid was characterized by development of multifocal suppurative dermatitis and suppurative bronchopneumonia. Localization of FISH-positive bacteria in necrotic lesions was restricted to the periphery. F. necrophorum would seem to have been unrecognized previously as a cause of abortion. The value of culture-independent diagnostic methods is emphasized.

Natural and experimental infection of sheep with European bat lyssavirus type-1 of Danish bat origin.

In 1998 and 2002, European bat lyssavirus type-1 (EBLV-1) was demonstrated in brain tissue of five Danish sheep suffering from neurological disorders. Four of the five sheep also had encephalic listeriosis. The animals originated from four flocks on pastures within a limited area of western Jutland. In a serological investigation in two of the herds, from which three of the diseased animals originated, EBLV-1 neutralizing antibodies were detected in only one of 69 sheep. In follow-up surveys, 2110 sheep sera collected at Danish slaughterhouses during 2000 were all negative for EBLV-1-antibodies, and EBLV-1 was not demonstrated in 87 ruminants displaying neurological symptoms. To investigate the pathogenic effects of EBLV-1, four sheep were inoculated intralabially with either brain material from one of the naturally infected sheep or virus isolated from the same sheep. These animals developed EBLV-1 neutralizing antibodies at 5-9 weeks post-inoculation but did not exhibit neurological signs during a 33-week observation period. It was speculated that the immune response prevented viral dissemination to the brain, resulting in an abortive peripheral infection. It was concluded that EBLV-1 can infect sheep under natural conditions as an incidental event.

Renal lipofuscinosis in Danish slaughter cattle.

A study was performed to characterize dark brown or black discoloured kidneys ("black kidneys") in Danish slaughter cattle and to investigate the aetiology and pathogenesis. In 133 939 cattle entering four abattoirs, 359 cases of "black kidneys" were recorded. Of these, 57 cases were submitted for macroscopical, microscopical, and ultrastructural examination. A pigment with characteristics similar to those of lipofuscin was found in secondary lysosomes in epithelial cells of the proximal tubules. Pigment accumulation was the cause of discoloration, with a positive correlation between the discoloration of the renal cortex and the degree of pigment accumulation. Cases occurred only in cattle of the Holstein breed or the Red Danish Dairy breed and mainly in animals aged 3 years or older. In these breeds, prevalences of 0.44% and 2.51% were found, respectively. Epidemiological analyses indicated that affected animals aged 4.5 to 6.5 years or 7.5 to 8.5 years were culled more frequently than unaffected cattle. Epidemiological and genealogical analyses strongly indicated a genetic aetiology with simple autosomal recessive inheritance.