INTRODUCTION

Calcigenic plants are among the most harmful plants for animals in the world (Odriozola ^{et al., 2018}; ^{Zanuzzi et al., 2008}), the chemical nature of the toxic agents contained induce calcinosis, the main active principle is a steroid glycoside that is hydrolyzed in the intestine, rumen and other tissues and releases the steroid fragment, which is in most cases 1,25 (OH)₂D₃ (^{Waser et al., 1983}; ^{Dallorso et al., 2001}; ^{Wu and Sun, 2011}). Excess vitamin D stimulates calcium binding protein (CaBP) synthesis and calcium and phosphate absorption, producing hypercalcemia and hyperphosphatemia (^{Sun, 2010}; ^{Dirksen et al., 2003}). The excess absorbed mineral cannot be metabolized, is deposited in soft tissues and produces calcinosis (^{Mello, 2003}; ^{Zanuzzi et al., 2012}).

Calcinosis caused by chronic vegetable poisoning, is a well-known disease in Argentina, Brazil, Paraguay and Uruguay, has received different names, such as “enteque ossificans”, “bichoquera”, “espichamiento” and “enteque seco” (^{Odriozola et al., 2018}; ^{Machado et al., 2020}). Similar pictures have been described in various regions of the world affecting animals in grazing. In all of them, the deposition of calcium salts in soft tissues, is accompanied by a severe physical deterioration and decay of animals. So far, six plants that induce systemic calcinosis have been described by chronic poisoning: Cestrum diurnum, Nierembergia veitchii, Solanum glaucophyllum, Solanum torvum, Stenotaphrum secundatum and Trisetum flavescens ( ^{Grabner et al., 1985}; ^{Braun et al., 2000}).

Trisetum flavescens causes an enzootic calcinosis in Germany and Austria, as well as presenting in South America. The ingestion of Cestrum diurnum affects bovine and equine livestock in Florida, USA. Solanum trovum has been associated with cattle in Papua, New Guinea. Cestrum diurnum has been associated as the disease cause in Hawaii and Jamaica. Solanum sodomaeum has been mentioned in disease processes in Hawaii. In Brazil it has been recognized since 1968 and described on farms of the Julio de Castihos municipality, Rio Grande do Sul; and the disease has been reproduced experimentally in rabbits and sheep through the administration of Nierembergia veitchii (^{Zanuzzi et al., 2008}; ^{Zanuzzi et al., 2012}; ^{García et al., 2012}; ^{Rissi et al., 2009}).

Trisetum flavescens is a vegetable that can be ingested in any state, even being dry, so it can be a cause of poisoning for the animals at grazing time. The table is characterized by calcinosis, with calcium deposition in soft tissues, as is the case of muscles and tendons, heart and large arteries, including aorta (^{Jennings, 1969}; ^{Cuesta, 2003}).

This study describes the clinical case of a sheep that, according to background and data provided by the owner, had been ingesting Trisetum flavescens, and showed clinical signs of decay, among others; the most evident lesions in the macro and microscopic study were associated with cartilaginous metaplasia of the right atrium, a condition that highlights the risk of consuming plants, which may contain a variety of substances that can affect and deteriorate the health of different herbivorous domestic species.

METHOD

Whole blood was collected from the sheep in tubes without anticoagulant and sacrificed according to ^{SAGARPA (1995).} An anatomopathological study was carried out to evaluate macroscopic changes; samples of atria and heart among other tissues were collected and fixed in 10% buffered formalin (10:1); hematoxylin-eosin staining was used for histological evaluation.

Plant samples were sent to the herbarium of the National Autonomous University of Mexico for taxonomic identification.

RESULTS

Anatomopathologic study

Relevant macroscopic findings: in the heart there was mucoid degeneration of the coronary fat and the right atrium presented hard nodulations of whitish color to the touch (Figure 1 and 2). Pleural adhesions, hypostatic congestion, hemoperitoneum, reticulum, rumen, omasum and abomasum distention were present. The presumptive diagnosis was cartilaginous metaplasia of the right atrium.

Figure 1 Pericardium cut. Light hydropericardium, thickened pericardium and opaque appearance. 

Figure 2 Heart. Rounded contour, with mucooid degeneration of fat, whitish areas in the right atrium (hard to tact and cutting). 

Histopathological study

The most significant finding in the microscopic study of the right atrium was cartilaginous metaplasia where hyaline cartilage and areas of myocardial ossification were observed at the atrium edges (Figures 3 and 4).

Figure 3 Histological cutting of heart wall. Tumefaction and undulation of muscle fibers, presence of Sarcocystis; replacement of myocytes by chondrocytes in cardiac muscle. H&E stain. 40X. 

Figure 4 Histological court of right atrium. A. Chondrocytes that have replaced the cardiac muscle. 4x B. Gaps occupied by hypertrophied chondrocytes (relief image). 100X.H&E stain. 

A whole blood sample without anticoagulant was sent for complementary clinical biochemistry testing, which indicated hyperphosphatemia and hypermagnesemia (Table 1).

Table 1 Sheep clinical biochemistry results 

Analyte	Value obtained.	Reference value
Calcium	2.2 mmol/l	2.25-2.6 mmol/l
Phosphorus	7.51 mmol/l	1.40-2.40 mmol/l
Magnesium	1.29 mmol/l	0.9-1.14 mmol/l

DISCUSSION

The most relevant finding of the clinical pathological study in the sheep was what was observed in the heart, which led to the definitive diagnosis of atrial cartilaginous metaplasia associated with the ingestion of Trisetum flavenscens, better known as "golden oats", a plant that is toxic to cattle; this was reaffirmed and considered in the clinical history detected in the auscultation, process presentation form and by the data of plant consumption by the animal. ^{Dirksen et al. (2003)} , when evaluating different sheep of a flock that remained in a pasture consuming Trisetum flavenscens, observed in several sheep the typical lameness of calcinosis (slightly bent carpus and relatively stretched position of the tarsal and spur joints when standing, "kneeling" in the carpus), with a progressive deterioration of the circulatory and respiratory systems (holosystolic endocardial murmur, congestion and expiratory dyspnea). In contrast, it is worth mentioning that as the only pathological case that occurred in the sheep production unit, the MVZ attending these animals, reported that on cardiac auscultation he heard a faint gallop noise.

The initial cartilaginous metaplasia of myocardium associated with the consumption of Trisetum flavenscens may have and show variable signs and injuries, as ^{Dirksen et al (2003)}, ^{Reilly et al. (2012}) and ^{Scott (2007)}, which consider the cartilaginous metaplasia of cardiac valves, endocardium and arterial vessels in most cases are serious; and that to the pathomorphological evaluation are consistent with cardiovascular insufficiency (^{Cebra and Cebra, 2012}; ^{Brounts et al., 2005}) Other relevant findings are the calcification of several tendons and ligaments, kidneys and in chronic cases of the pulmonary parenchyma (^{Ribeiro et al., 2017}; ^{Estepa et al., 2006}).

When considering the information provided by the owner that the animal consumed Trisetum flavescens, it was related to an intoxication process, although it was not possible to determine the amount of ingestion per day, at least it had been consuming the plant for about a month and a half to two months. In the necropsy evaluation of the sheep in this study, no other significant lesions were observed in other tissues, only what was found in the heart; which is also relevant due to the few reports of similar cases. The concentration of vitamin D in Trisetum flavescens is 600 to 800 IU kg/MS. ^{Mello (2003)} and ^{Jennings (1969)}, refer that, with biochemical alterations, morphological changes are consequently presented, mainly calcifications in elastic fibers and basal membranes of those tissues with a tendency to calcify (heart, arteries and kidneys among other tissues).

Through the biochemistry performed, hyperphosphatemia and hypermagnesemia are relevant data to consider in the diagnostic orientation of the case; this type analyte imbalance can cause specific disorders in the metabolism of physiological importance compounds, such as calcium, the consumption periodicity of the identified vegetable would determine the signology and pathology. Furthermore, the individual variation of each organism in response to the phosphorus-calcium-magnesium imbalance (and possibly vitamin D and K) should be considered, since according to their mobility or deposition they will infer in the imbalance or functional alteration as in the analyzed case. In the information referred to by ^{Scott (2007)}, it mentioned that serum calcium and phosphorus concentrations increase between 20 and 25%, with increases of up to 3.4 mmol/l of calcium and 4 mmol/l of phosphorus; and given the frequency of this type of problem, it suggests radiological evaluation, where tissue calcification can be detected.

Grazing animals in problem areas present hypercalcemia and hyperphosphatemia, the "in situ" expression of osteopontin seems to be of particular importance in the genesis of calcifications since this protein binds very strongly to hydroxyapatite and therefore plays an important role in the calcification process, the disease makes its appearance in summer and autumn, especially in times of drought (^{Mello, 2003}; ^{Reilly et al., 2012}).

Affected animals move reluctantly, with short and stiff steps, sick animals show dyspnea and tachycardia when moved, and may fall to the ground with signs of cardiac and pulmonary insufficiency. When getting up, they do it with difficulty in subclinical cases; for its correct differential diagnosis, it could be useful to determine serum calcium and phosphorus values for the determination of osteocalcin, osteopontin and other proteins induced by the vitamin D effect in cellular differentiation. Only 12 weekly grams of Trisetum flavescens leaves are enough to reproduce the disease in a 300 kg cow. In 4 months, a bovine that ingested 50 leaves per day became clinically ill in 8 to 10 weeks (^{Cuesta, 2003}; ^{Machado et al., 2020}).

This problem has been present worldwide for several years, affecting the American continent first and foremost. The enzootic calcinosis of Central Europe is caused by chronic intoxication by the grass known as golden oats (Trisetum flavescens), considered until a few years ago as a valuable forage crop. The other plants of recognized calcinogenic capacity are Cestrum diurnum, Solanum torvum, Nierembergia veitchii and Stenotaphrum secundatum cause the disease in limited areas (^{Garcia et al., 2012}; ^{Gupta, 2012}).

In Germany and Austria, bovine enzootic calcinosis is caused by golden oats (Trisetum flavescens) that grows in the Alpine area and is much less toxic than white peak (S. Malacoxylon), since it has to integrate pastures into 20- 25% to cause toxicity (^{Waser et al., 1983}; ^{Dallorso et al., 2001}; ^{Zanuzzi et al., 2012}; ^{Braun et al., 2000}; ^{Gupta, 2012}).

According to the valuation of ^{Rissi et al. (2009)}, they mean that enzootic calcinesis of pets has been described in various parts of the world. Various plants have these calcinogenic properties. In bovines it has been shown that the disease is produced by the ingestion of Solanum malacoxylon in Argentina, Brazil and Uruguay; while in Cuba the Cestrum diurnum is disseminated throughout the island, which causes the disease in drought season.

While the presentation of enzootic calcinosis in South America, it coincides with the Trisetum Flavescens distribution area is a very serious problem in the basin of del Río Salado in Buenos Aires, but it is present throughout La Plata basin. The incidence of this disease is very difficult to estimate due to the existence of numerous subclinical cases, has been estimated at 10% in Buenos Aires and by 8.2% in Santa Fe ( ^{Zanuzzi et al., 2012}; ^{Rissi et al., 2009}).

The wide regions of animal production in Mexico are conducive to plant diversity development that grow naturally, which may contain a variety of substances that must be determined and evaluated, in order to minimize the occurrence of intoxication cases in different herbivorous domestic species, with a consequent morphological and functional alteration as the one referred to in this study.

CONCLUSION

The right atrium cartilaginous metaplasia observed in the sheep was associated with the high consumption of the toxic plant "golden oats" (Trisetum flavescens), which can grow in different lands; and be a toxicosis risk for different animal species and cause significant alterations and even death.