Enzootic calcinosis in Toggenburg goats in New Zealand.

CASE HISTORY: Necropsies on Toggenburg goats culled from a small farm in the Manawatu district of New Zealand, performed at Massey University (Palmerston North, NZ) over a period of 29 years (1991-2019), revealed soft tissue mineralisation, particularly of cardiovascular tissues. The farm spans 10 acres and runs between 15 and 30 Toggenburg goats. The goats are predominantly on pasture comprising a variety of types. PATHOLOGICAL FINDINGS: Necropsies were performed on all adult goats (n = 45) that died or were euthanised. Histopathology was performed on 42 goats (93%), of which 33 (73%) included sufficient tissues diagnostically relevant to soft tissue mineralisation. The most significant gross findings were in various arteries, with the aorta most commonly affected, followed by the heart and lungs. The aortic intima showed prominent, multifocal to coalescing, raised, wrinkled, white plaques. Microscopically there were multiphasic lesions of mineralisation, chondroid, and osseous metaplasia in the elastic arteries, aorta, heart and lungs. A lumbar vertebra from one goat had prominent, basophilic, fibrillar, tangled matrix lining Haversian canals and lamellae. LABORATORY FINDINGS: Blood samples were collected from 15 adult goats in the affected herd and from 10 adult Toggenburg goats from an unaffected herd. Samples were collected by jugular venipuncture at 2-month intervals for 12 months (April 2018-March 2019). Concentrations of calcium, phosphorus, 25-hydroxyvitamin D2 and D3 (25OHD2, 25OHD3) in serum were analysed. The concentration of total 25OHD in serum was 34.2 (95% CI = 18.9-49.4) nmol/L (p < 0.001) higher in goats from the affected herd than in goats from the unaffected herd. Serum 25OHD2 concentration was 46.2 (95% CI = 39.2-53.2) nmol/L higher (p < 0.001) in goats from the affected herd compared to the unaffected herd. Serum Ca concentrations in affected goats were 0.101 (95% CI = 0.005-0.196) mmol/L higher (p = 0.039) than unaffected goats, but remained within the reference range. There was no evidence of a difference in serum 25OHD3 and P concentration between the herds. VEGETATION SURVEY: All paddocks on the property were surveyed every 2 months along evenly spaced line transects, and then further traversed perpendicularly to form a grid. No known calcinogenic species were identified. Known plant sources of vitamin D identified on the farm included mushrooms (species not defined), Dactylis glomerata, lichen, pine pollen, and algae. DIAGNOSIS: Soft tissue mineralisation and enzootic calcinosis. CLINICAL RELEVANCE: Veterinarians are alerted to the possibility of either enzootic calcinosis in goats and the potential occurrence of calcinogenic plants in New Zealand; or chronic vitamin D toxicosis of non-plant origin.

Poisoning by Nierembergia veitchii: Effects on vascular smooth muscle cells in the pathogenesis of enzootic calcinosis.

Vascular mineralization is a hallmark of enzootic calcinosis. Histopathological, ultrastructural, and immunohistochemical investigations were performed on the external carotid arteries of seven sheep naturally poisoned by Nierembergia veitchii. Histologically, moderate to marked hyperplasia of the tunica intima was observed without mineralization. The tunica media exhibited mild to severe mineralization and osteochondroid metaplasia. Sheep with enzootic calcinosis showed arterial overexpression of osteopontin and tissue-nonspecific alkaline phosphatase and immunolabeling for osteonectin and osteocalcin in both intima and media layers of the tested arteries. The main ultrastructural finding in the tunica media was a marked phenotypic change of vascular smooth muscle cells from a contractile phenotype (VSMC-C) into a synthetic phenotype (VSMC-S). In the tunica media, VSMC-S produced matrix and extracellular vesicles, forming mineralizable granules associated with arterial mineralization. VSMC-S were also present in the tunica intima, but matrix and extracellular vesicles and mineralization were not observed. The absence of matrix and extracellular vesicles in the intimal hyperplasia, even in the presence of noncollagenous bone proteins, tissue-nonspecific alkaline phosphatase, and vitamin D receptors, reinforces the hypothesis that the presence of matrix and extracellular vesicles are crucial for the development of vascular mineralization in enzootic calcinosis. It is proposed that the two different VSMC-S phenotypes in calcinosis are due to the expression of at least two genetically different types of these cells induced by the action of 1,25(OH)2D3.

The Solanum glaucophyllum Desf. extract reduces mineralized matrix synthesis in osteogenically differentiated rat mesenchymal stem cells in vitro.

The Solanum glaucophyllum Desf. has been used to treat and prevent diseases in human and veterinary medicine. On the other hand, plant poisoning causes several bone diseases, among them osteoporosis, which is characterized by osteoblastic hypoplasia. Because the osteoblast is a cell derived from the differentiation of mesenchymal stem cells (MSCs) from bone marrow, the hypothesis is that the plant reduces the osteogenic differentiation of MSCs. The objective of this study was to evaluate the effects of S. glaucophyllum Desf. extract on MSCs cultured in osteogenic differentiation medium. We determined by liquid chromatography that 1 ml of plant extract contained 3.8 µl of 1,25(OH)2 D3 (calcitriol). Four groups of MSCs cultivated in osteogenic medium were evaluated as follows: (a) treated with 100 µl of extract/L containing 0.4 µg/L of calcitriol; (b) treated with 1 ml of extract/L containing 4 µg/L of calcitriol; (c) treated with 5 ml of extract/L containing 20 µg/L of calcitriol; and (d) a control group without extract. We performed alkaline phosphatase activity assay, analysis of MTT conversion to formazan, and evaluated the percentage of cells, and number and diameter of mineralization nodules. The expression of gene transcripts for osteopontin, bone sialoprotein and BMP-2 was analysed by RT-qPCR. After 21 days, there was a significant reduction in MTT conversion to formazan in treated groups, of the cellularity in the group with 5 ml of extract/L, and in the number and size of mineralization nodules in the groups treated with 1 and 5 ml of extract/L. The 5 ml extract/L concentration also reduced transcript expression of osteopontin. It is concluded that S. glaucophyllum Desf. at concentrations of 1 and 5 ml extract/L reduced mineralized matrix synthesis in MSCs cultivated in osteogenic differentiation medium, which suggests that this is one of the mechanisms by which osteoporosis occurs in intoxicated animals.

Ultrasonographic findings in two sheep with enzootic calcinosis.

INTRODUCTION: This report describes 2 sheep with enzootic calcinosis characterized by abnormal cardiovascular and respiratory findings and ascites causing abdominal distension. Both sheep were anorexic and listless and had increased heart and respiratory rates. Auscultation of the heart revealed a gallop rhythm in sheep 1 and a loud systolic heart murmur in sheep 2. The activities of liver enzymes were severely increased in both sheep. Abdominal ultrasonography showed severe ascites and congestion of the liver and caudal vena cava. Echocardiography in sheep 2 showed hyperechoic and markedly thickened mitral and aortic valves with moderate-severe mitral insufficiency and generalized cardiomegaly. Both sheep were euthanized and examined postmortem. In addition to ascites and pleural effusion, the principal lesions were nodular thickening of the heart valves and calcification of the aorta and other arteries. Nutrition of the sheep did not include hay pellets, but the sheep were kept together with alpacas and lamas and had access to the hay pellets of these animals. In addition visitors were allowed to feed the sheep with hay pellets offered by the zoo in a dispenser. The two types of hay pellets had Vit D concentrations of 9'900 IU VitD3/kg and 7'000 IU Vit D2/kg, respectively. The definitive diagnosis was enzootic calcinosis.

Nierembergia rivularis poisoning in cattle.

Nierembergia rivularis causes enzootic calcinosis (EC) in sheep. In this work, we describe EC caused by N. rivularis in cattle. For 3 years cattle grazing in 7 paddocks were evaluated. Cows with clinical signs compatible with EC were detected in only one paddock with a morbidity of 9.4%, 24.5%, and 34.5% during the summer of 2019, 2020, and 2021, respectively. Affected cows weighed 55 kg less (p < 0.01) than cows without signs of the same paddock, and 19.6% of these cows had hypercalcemia and/or hyperphosphatemia. Typical soft tissue calcification was observed in 3 autopsied cows. Additionally to the arterial calcification, 2 cows had multiple mineralized foci in several veins. In the Paddock A where EC occurred, the pasture contained 7-12% N. rivularis. In the other 6 paddocks (Paddocks B-G) where EC had not occurred, the pasture had 0.2-3.5% N. rivularis. Cows grazing in Paddock A had ∼30% lower pregnancy rates than cows from Paddocks B-G. At the slaughterhouse, the carcasses of 45 cows from Paddock A weighed 17.6% (p < 0.01) less than 93 carcasses of cows from Paddocks B-G. Furthermore, the carcasses of cows from Paddock A were classified as low quality. Eight cows with EC signs from Paddock A and 10 cows without EC signs from Paddocks B-G were removed to a Lolium multiflorum pasture. After 120 days of grazing, the cows from Paddock A gained 45.2% less (p < 0.01) live weight than cows from Paddocks B-G. Poisoning with N. rivularis may cause significant economic losses in Uruguay due to low fertility rates and weight gain of affected cattle.

Enzootic calcinosis in ruminants: A review.

Enzootic calcinosis (EC), is a chronic disease, caused by toxic plants, which has mainly affected ruminants for more than 100 years. It is characterized by a soft tissue calcification, hypercalcemia, hyperphosphatemia, hypoparathiroidism, hypercalcitoninism, osteonecrosis, and osteopetrosis. The toxic compound reported in some of these plants are 1,25(OH)2D3 glycosides. Characterization of calcinogenic principles were performed with Solanum glaucophyllum, Trisetum flavescens, Cestrum diurnum, and Stenotaphrum secundatum. Other plant species involved in EC are: Nierembergia rivularis, Nierembergia veitchii, Solanum torvum, and Solanum stuckertii. Clinical and pathological findings are well characterized. We present different aspects of the pathogenic mechanism of EC, including genetic modulation, expression of bone proteins, cellular de-differentiation of arterial smooth muscle cells, phenotypic changes, production of a calcifiable matrix, and modulation of the calcium and phosphorus balance. The use of comparative pathology can bring significant advances in the understanding of the pathogenic mechanism. In this review we present a broad approach to the different aspects of intoxication: history of EC on different continents; plant species, distribution and morphology; toxic principle; pathogenesis; epidemiology; clinical signs; diagnosis; and control and prophylaxis.

Rat mesenchymal stem cell cultures as a model to elucidate the cellular and molecular pathogenesis of bone metaplasia induced by Solanum glaucophyllum intoxication.

The hypothesis of this experiment is that mesenchymal stem cells (MSCs) are involved in the genesis of the bone metaplasia caused by Solanum glaucophyllum intoxication. We determined using liquid chromatography that 1 mL of plant extract contained 3.8 µl of 1,25(OH)2D3. The ability of 100 µL, 1 mL and 5 mL of extract/L, containing 1 nM (0.4 µg/L), 10 nM (4 µg/L) and 50 nM (20 µg/L) of 1,25(OH)2D3, respectively, in inducing the osteogenic differentiation in bone marrow MSCs from rats was tested. At the concentrations of 1 and 5 mL of extract/L of culture medium without osteogenesis-inducing factors, the plant extract induced the osteogenic differentiation of the MSCs, as was evidenced by the greater synthesis of mineralized matrix. At the higher concentration (5 mL of extract/L), an increase in the relative expression of BMP-2 gene was observed. It was concluded that rat bone marrow MSC culture is a good model for studying the effects of the S. glaucophyllum extract on the osteogenic differentiation of undifferentiated cells. Also, S. glaucophyllum extracts containing 10 nM (4 µg/L) and 50 nM (20 µg/L) of 1,25(OH)2D3 induce the osteogenic differentiation of MSCs, suggesting that this is one of the mechanisms by which S. glaucophyllum causes bone metaplasia.

Enzootic calcinosis in horses grazing Solanum glaucophyllum in Argentina.

Solanum glaucophyllum, a toxic plant known for its calcinogenic effects, causes enzootic calcinosis in ruminant and monogastric animals. We describe an outbreak of enzootic calcinosis that occurred in a herd of 110 horses grazing pastureland heavily contaminated with S. glaucophyllum in Buenos Aires province, Argentina. Ten horses developed clinical signs, and 6 horses died. Clinical signs included abnormal gait (stiff-legged action, short strides), stiffness, thoracolumbar kyphosis, reluctance to move, wide stance, chronic weight loss, weakness, recumbency, and difficulty standing. Autopsy of 2 horses revealed severe mineralization of the aorta, pulmonary arteries, heart, and lungs, consistent with enzootic calcinosis. Although horses usually have very selective grazing behavior, under food restriction conditions, they can ingest the toxic plants and can develop the disease. Enzootic calcinosis should be considered as a differential diagnosis in horses grazing S. glaucophyllum-invaded pasturelands with compatible clinical signs and lesions.

Enzootic calcinosis in sheep in Uruguay: a brief review and report of two outbreaks / Calcinose enzoótica em ovinos no Uruguai: revisão de literatura e descrição de dois surtos

This study aimed to do a brief review of enzootic calcinosis in sheep and to report two outbreaks of Nierembergia rivularis poisoning in sheep in Uruguay. The outbreaks occurred in farms located on an island (Outbreak A) and on the border (Outbreak B) of the Rincón del Bonete lake. Sheep of all ages were affected, with the exception of suckling lambs. The first clinical signs occurred in early October, and deaths occurred from December to February. Outbreaks A and B had morbidity of 10%, and the mortality was 7.2% and 2.8% in Outbreaks A and B, respectively. The clinical signs included weight loss, retracted abdomen, stiff gait, and kyphosis. An autopsy was performed on one sheep from each outbreak. Pulmonary and arterial calcification, nephrocalcinosis, and osteopetrosis were observed in gross and microscopic examination in both sheep. Thyroid C-cell hyperplasia and carcinoma was observed in sheep A. Sheep B showed thyroid C-cell hyperplasia and parathyroid chief cell atrophy. The parathyroid was not examined in the sheep from Outbreak A. The differential diagnosis of enzootic calcinosis in southern South America should consider four toxic plants in the Solanaceae family: Solanum glaucophyllum, Solanum stuckertii, Nierembergia veitchii, and Nierembergia rivularis.(AU)

Este trabalho faz uma breve revisão da calcinose enzoótica em ovinos e descreve dois surtos de intoxicação por Nierembergia rivularis em ovinos no Uruguai. Os surtos ocorreram em propriedades localizadas em uma ilha (Surto A), e nas margens (Surto B) do lago do Rincón del Bonete. Foram afetados ovinos de todas as idades, exceto cordeiros lactentes. Os primeiros sinais clínicos ocorreram no início de outubro e as mortes de dezembro a fevereiro. Morbidade de 10% foi observada nos Surtos A e B. A mortalidade foi de 7,2% e 2,8% nos Surtos A e B, respectivamente. Os sinais clínicos incluíram perda de peso, abdômen retraído, marcha rígida e cifose. Foram necropsiados um animal de cada rebanho. Observou-se mineralização arterial e pulmonar, nefrocalcinose e osteopetrose no exame macroscópico e histológico dos dois ovinos. Hiperplasia e carcinoma de células C da tireoide foram observados no ovino A. O ovino B apresentou hiperplasia de células C da tireoide e atrofia das células principais da paratireoide. As paratireoides do ovino A não foram examinadas. O diagnóstico diferencial da calcinose enzoótica no Sul da América do Sul deve considerar quatro plantas calcinogênicas da família Solanaceae: Solanum glaucophyllum, Solanum stuckertii, Nierembergia veitchii e Nierembergia rivularis.(AU)

Calcinose enzoótica em ruminantes no Brasil Central / Enzootic calcinosis in ruminants in Central Brazil

A calcinose enzoótica é uma enfermidade caracterizada por mineralização de artérias e tecidos moles, osteopetrose, hipercalcemia e hiperfosfatemia. Neste trabalho descreve-se uma calcinose enzoótica em ruminantes no Mato Grosso, Goiás, Minas Gerais, Tocantins e no Distrito Federal, em campos onde não foi encontrada nenhuma das plantas calcinogênicas conhecidas. Os primeiros casos foram diagnosticados em 2004, e até 2010 foram necropsiados 86 casos da doença provenientes de 42 propriedades. Trinta e três propriedades foram visitadas, e em 32 os pastos eram caracterizados por moderada a acentuada degradação e invasão por plantas daninhas. A doença foi diagnosticada em ovinos em 19 fazendas, em bovinos em 17, em caprinos em 5 e em uma fazenda foram afetados tanto caprinos quanto ovinos. Animais adultos foram mais acometidos, mas a doença foi observada, também, em animais lactentes. A enfermidade foi observada durante todo o ano, mas a maioria dos surtos ocorreu nos meses de maior índice pluviométrico. Em duas fazendas foram determinados, mensalmente, os níveis séricos de Ca e P observando-se uma elevação significativa dos mesmos durante o período de chuvas. Os principais sinais clínicos observados foram de emagrecimento progressivo, caquexia e flexão de membros anteriores, com andar rígido. As principais lesões macroscópicas e histológicas foram de mineralização das artérias, valvas cardíacas, pulmões e rins. Sugere-se que a doença é provocada por uma planta calcinogênica ainda desconhecida. Mesmo sem conhecer a causa da calcinose recomenda-se evitar o pastejo de ruminantes em áreas degradadas e reformar pastagens degradadas.

Enzootic calcinosis is a disease characterized by mineralization of soft tissues, osteopetrosis, hypercalcemia and hyperphosphatemia. From 2004 to 2010, 86 cases of enzootic calcinosis were diagnosed in 42 farms in the states of Mato Grosso, Goias, Minas Gerais and Tocantins, and in the Federal District. The pastures were inspected in 33 farms, observing that in 32 outbreaks the disease occurred in degraded pastures invaded with different weeds. Previously known calcinogenic plants were not present. The disease was diagnosed in sheep in 19 farms, in cattle in 17 farms, in goats in five farms, and in sheep and goats in one farm. Adult animals were more affected; but the disease also was observed in lactating animals. The disease occurred all over the year, but most outbreaks were diagnosed during the rainy season. Serum Ca and P concentrations determined monthly in two sheep flocks were significantly higher during the rainy season. Main clinical signs were progressive emaciation followed by cachexia, and stiff gait with flexion of the fore limbs. Gross findings were mineralization of the arteries, cardiac valves, lungs, and kidneys. Histological findings were arterial mineralization, mainly in the medial layer with cartilaginous metaplasia and colagenous deposition between smooth muscle fibers. It is suggested that the disease is caused by an unknown calcinogenic weed present in degraded pastures.