CASE NOTES

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Toxic nephropathy in cattle grazing pastures heavily infested with blue heliotrope (Heliotropium ampexicaule)

Bruce Watt, Central Tablelands Local Land Services, Bathurst; Michael Willott, Upper Macquarie County Council, Bathurst; Patrick Staples and Erika Bunker, Veterinary Pathologists, NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle

Posted Flock and Herd August 2024

Introduction

Common heliotrope (Heliotropium europium) and blue heliotrope (Heliotropium ampexicaule) are introduced weeds that contain pyrrolizidine alkaloids (PAs) known to cause disease, mostly of the liver, in several species of livestock. Pigs and chickens are highly susceptible, cattle and horses moderately susceptible, and sheep and goats relatively resistant. All above-ground parts and seeds of heliotropes are toxic (McKenzie 2012).

Blue heliotrope was introduced to Australia from South America in the late 19^th century and is now a widespread major agricultural weed in NSW. It is drought-hardy and adaptable to a wide range of soil and climate types. It spreads aggressively via seeds as well as root fragments (McCafferty et al. 2008). It is a declared noxious weed in many government areas of NSW. Control is difficult. Available control measures include pasture and grazing management, herbicides and biological agents (Briese 2012).

Image 1. Blue Heliotrope flowers. Photo: J Edwards

History

In April 2021, the owner of a property in the NSW Central Tablelands reported that one of 40 mature Angus cattle with about five-month-old calves at foot died after becoming emaciated and that four other cows were in poor to emaciated condition while the remainder of the herd was in fat condition. The cows were running on native, red-grass dominant (Bothriochloa macra) pastures with a major infestation of blue heliotrope (H. amplexicaule) and occasional rock fern (Cheilanthes sieberi). The identity of the plant was confirmed as Heliotropium ampexicaule (Vahle - det. AE Orme, 25 May 2023). The cows had been treated with moxidectin in December 2019, and recently received mineral supplements including selenium.

The owner reported that an occasional cow became emaciated and died each year prior to the breaking of the drought in March 2020 but after this time more cows were affected. The affected cows, mostly at least five years of age, lost weight over 6-12 months. There was no seasonal pattern to the deaths

Clinical examination

Four mature Angus cows (Cases 1-4) were examined and blood sampled on 27 April 2021. All cows had normal body temperatures, normal externally palpable lymph nodes, normal rumen motility with no abnormalities detected on chest auscultation and no oral or coronary band lesions. There was no evidence of diarrhoea. Case 1, in fat score 1.0, had crusty skin lesions over the head, neck and shoulders. Case 2 was in fat score 2.5 with normal mucous membranes and no sub-mandibular oedema. Case 3 was in fat score 1.5 with normal mucous membranes but submandibular oedema. Case 4 was in fat score 2 and did not have submandibular oedema but had slightly pale icteric mucus membranes.

Image 2. Two of the affected cows including Case 1 closest to the camera. Photo: Bruce Watt

Clinical pathology

Blood test results 27 April 2021

Table 1. Biochemistry

BIOCHEMISTRY
TEST	NORMALS	UNITS	RESULTS
SAMPLE			1	2	3	4	POOL
GGT	0-35	U/L	23	168 H	46 H	83 H
GLDH	0-30	U/L	97 H	149 H	190 H	87 H
AST	0-120	U/L	199 H	246 H	247 H	141 H
BIL	0.0-24.0	µmol/L	7.0	12.0	10.0	9.6
CK	0-300	U/L	304 H	468 H	228	139
UREA	2.1-10.7	mmol/L	9.5	14.8 H	11.9 H	16.4 H
CREAT	0-186	µmol/L	270 H	445 H	336 H	483 H
PHOS	0.80-2.80	mmol/L	3.75 H	3.91 H	3.39 H	3.79 H
URE/CREA	0.00-0.07		0.04	0.03	0.04	0.03
PROTEIN	60.0-85.0	g/L	55.8 L	59.0 L	54.5 L	72.6
ALBUMIN	25.0-38.0	g/L	26.5	26.2	23.0 L	29.6
GLOB	30.0-45.0	g/L	29.3 L	32.8	31.5	43.0
ALB/GLOB	0.7-1.1		0.9	0.8	0.7	0.7
BHB	0.00-0.80	mmol/L	0.28	0.18	0.21	0.19
CA	2.00-2.75	mmol/L	2.02	2.18	1.96 L	2.02
MG	0.74-1.44	mmol/L	0.67 L	0.85	0.83	0.91
GSH PX	40-300	U/gHb	34 L	19 L	26 L	21 L
PEPSIN	0.0-5.0	U/L	21.5 H	13.6 H	29.3 H	18.6 H
HAPTO	0.00-0.30	g/L	0.32 H	0.34 H	0.20	0.40 H
SERUM HB	0.00-0.20	g/dL	0.08	0.09	0.10	0.30 H
VIT.B12	200-500	pmol/L					704 H

Table 2. Haemotology

HAEMATOLOGY
TEST	NORMALS	UNITS	RESULTS
SAMPLE			1	2	3	4
PROT-RTS	65-85	g/L	61 L	70	63 L	79
FIBRIN.	3-7	g/L	5	7	7	7
PR/FI	15-100		11 L	9 L	8 L	10 L
PCV	23-44	%	26	24	22 L	22 L
RBC	5.00-8.00	1012/L	4.05 L	4.10 L	3.27 L	3.56 L
HB	8.0-15.0	g/dL	9.2	8.2	7.6 L	7.6 L
MCV	44-62	fL	64 H	59	67 H	62
MCHC	30-35	g/dL	35	34	35	35
MCH	14-20	pg	23 H	20	23 H	21 H
WBC	4.0-12.0	109/L	2.1 L	2.8 L	2.5 L	2.0 L
BAND N.	0.00-0.12	109/L	0.00	0.00	0.00	0.00
NEUTRO.	0.60-4.00	109/L	0.74	0.92	1.50	0.64
BAND/N	0.00-0.20		0.00	0.00	0.00	0.00
LYMPHO.	2.50-7.50	109/L	0.88 L	1.71 L	0.93 L	1.16 L
MONO.	0.03-0.84	109/L	0.21	0.03	0.05	0.12
EOSINO.	0.00-2.40	109/L	0.27	0.14	0.03	0.08
BASO.	0.00-0.20	109/L	0.00	0.00	0.00	0.00
PLATELET	100-800	109/L		120

There was a mild to moderate increase in GGT levels in three of four cows and a mild to moderate increase in GLDH levels in all four cows. Mild azotaemia and hyperphosphataemia were present in three cows and mild hyperphosphataemia was present in four cows. Three cows had mild hypoproteinaemia. Marginally low GSHPx levels were noted in all four cows. High pepsinogen levels in all cows were suggestive of moderate to major abomasal damage. Low normal (two cows) or marginally low (two cows) PCV was not accompanied by evidence of red blood cell regeneration. All four cows had lymphopaenia, a common finding in sick and stressed animals.

Pooled liver fluke serology suggested a high level of herd liver fluke infestation.

Laboratory test results suggested a possible hepatopathy, a probable nephropathy with elevated creatinine, urea and phosphorus in the absence of evidence of dehydration, possible parasitism (abomasal parasites, liver fluke) and marginal selenium status.

A follow-up visit was conducted on 12 May 2021 with the aim to determine the selenium and internal parasite status of five normal cohorts (Cases 5-9). Four faecal samples were collected, with average worm egg count of 220 epg (range 0-440). 85% were identified as Haemonchus sp. with 5% each of Trichostrongylus spp., Cooperia spp. and Ostertagia. No liver fluke eggs were detected. Bloods from five clinically normal cows (Cases 5-9) had similar marginal GSHPX levels as the affected cows (averaging 28 U/L range 16-35).

By early July, two of the four ill-thrift cows (Cases 1-4) had died, one (Case 1) had deteriorated and another cow appeared to have slightly improved. Bloods from both surviving cows, collected on 7 July 2021, revealed elevated urea and creatinine levels, presumably due to renal disease in the absence of dehydration. They also showed lymphopaenia, and one cow had moderately elevated GLDH and GGT levels.

Blood test results 7 July 2021

Table 3. Biochemistry

BIOCHEMISTRY
TEST	NORMALS	UNITS	RESULTS
SAMPLE			1	2
GGT	0-35	U/L	19	149 H
GLDH	0-30	U/L	14	95 H
AST	0-120	U/L	130 H	128 H
BIL	0.0-24.0	µmol/L	12.2	14.7
CK	0-300	U/L	1079 H	154
UREA	2.1-10.7	mmol/L	19.4 H	23.8 H
CREAT	0-186	µmol/L	261 H	627 H
PHOS	0.80-2.80	mmol/L	1.12	2.04
URE/CREA	0.00-0.07		0.07 H	0.04
PROTEIN	60.0-85.0	g/L	66.5	78.9
ALBUMIN	25.0-38.0	g/L	38.9 H	33.4
GLOB	30.0-45.0	g/L	27.6 L	45.5 H
ALB/GLOB	0.7-1.1		1.4 H	0.7
BHB	0.00-0.80	mmol/L	0.39	0.29
CA	2.00-2.75	mmol/L	2.49	2.29
MG	0.74-1.44	mmol/L	1.06	1.05
HAPTO	0.00-0.30	g/L	0.26	0.49 H
SERUM HB	0.00-0.20	g/dL	0.30 H	0.28 H

Table 4. Haemotology

HAEMATOLOGY
TEST	NORMALS	UNITS	RESULTS
PROT-RTS	65-85	g/L	75	92 H
FIBRIN.	3-7	g/L	3	7
PR/FI	15-100		24	12 L
PCV	23-44	%	32	27
RBC	5.00-8.00	1012/L	5.06	4.17 L
HB	8.0-15.0	g/dL	11.2	9.4
MCV	44-62	fL	63 H	65 H
MCHC	30-35	g/dL	35	35
MCH	14-20	pg	22 H	23 H
WBC	4.0-12.0	109/L	3.4 L	2.9 L
BAND N.	0.00-0.12	109/L	0.03	0.03
NEUTRO.	0.60-4.00	109/L	1.43	0.84
BAND/N	0.00-0.20		0.02	0.04
LYMPHO.	2.50-7.50	109/L	1.60 L	1.65 L
MONO.	0.03-0.84	109/L	0.24	0.17
EOSINO.	0.00-2.40	109/L	0.10	0.20
BASO.	0.00-0.20	109/L	0.00	0.00
NUCL.RBC		/100wbc	0	1
PLATELET	100-800	109/L	188	395

Gross pathology

On 7 July 2021 Case 1, being emaciated and weak with slightly pale mucous membranes, was euthanased. She had patches of scurfy skin lesions on the face and alopecic circular or annular lesions on the ventral abdomen. She was pregnant (mid-term). The liver was perhaps small with normal colour and relatively normal texture. The kidneys were possibly small, the cortex uniformly pale tan and the medulla relatively normal. About 100 ml of fine grit was noted in the abomasum, with coarse grit in the duodenum.

On 25 July another cow (Case 10) was autopsied after being found dead. She was in fat score 2 and the carcass was dehydrated. Subcutaneous tissues were yellow. The liver was yellow and appeared moderately autolysed. Kidneys appeared normal. The cow was approximately seven months pregnant.

Image 3. Kidney, Case 1, 7 July 2021. Photo: Bruce Watt

Image 4. Kidney closeup, Case 1, 7 July 2021. Photo: Bruce Watt

Image 5. Liver, Case 1, 7 July 2021. Photo: Bruce Watt

Histopathology

Case 1 had severe tubulointerstitial nephropathy, with tubular degeneration, atrophy and megalocytosis, and chronic fibrosing and lymphoplasmacytic interstitial nephritis. Tubuli often were either dilated and attenuated, or they were remnants of collapsed distorted tubuli with pleiomorphic, often large cells with large, sometimes bizarre nuclei, with occasional intranuclear cytoplasmic invaginations. The liver of this cow only showed mild changes consisting of scattered hepatocytes with mildly enlarged nuclei (karyomegaly).

In the liver of Case 10, hepatocytes showed marked anisocytosis and anisokaryosis, particularly in periportal areas, with a few large hepatocytes with large vesicular nuclei (borderline megalocytosis) and occasional binucleated cells. Mild fibrosis and mild biliary proliferation were noted in some but not all portal triads. This cow also had a distinct zonal pattern of severe periacinar fatty hepatocyte degeneration.

The renal changes in Case 10 were similar to those of Case 1, but interstitial fibrosis was milder and less widespread. Tubular injury was severe, with degeneration, necrosis and marked pleiomorphism of epithelial cells, with megalocytosis, occasional intranuclear cytoplasmic invaginations, binucleated cells and some bizarre cells. Often tubuli had transformed into misshapen remnants, with blurred borders between those and the interstitium.

Image 6. Kidney, Case 1, tubular dilation and attenuation, severe interstitial fibrosis with tubular remnants (H&E, 50x)

Image 7. Kidney, Case 1, tubular remnants with pleiomorphic large cells and intranuclear cytoplasmic invaginations (arrows) (H&E, 200x)

Image 8. Kidney, Case 1, megalocytosis of tubular epithelium with a sloughed intraluminal epithelial cell exhibiting karyomegaly (arrows) (H&E, 200x)

Image 9. Liver, Case 1, hepatocyte showing karyomegaly (arrow) (H&E, 200x)

Image 10. Liver, Case 10, anisocytosis and anisokaryosis with borderline megalocytosis (arrows) (H&E, 200x)

Image 11. Kidney, Case 10, tubular necrosis with bizarre megalocyte (H&E, 200x)

Image 12. Kidney, Case 10, tubular injury with megalocytosis (arrow) (H&E, 200x)

Image 13. Kidney, Case 10, tubular necrosis with a megalocyte with an intranuclear inclusion (H&E, 200x)

Image 14. Kidney, Case 10, megalocyte with vesicular nucleus (H&E, 200x)

Discussion

Renal failure was the most likely cause of illness and death in these cows, as supported by the severe renal lesions and azotaemia in several cows including the necropsied animals.

The cows had been grazing on blue heliotrope-dominant pasture, with no access to other known hepatotoxic or nephrotoxic plants.

Both the liver and kidneys had cellular changes that could be attributed to the effects of pyrrolizidine alkaloids (PAs), including megalocytosis, karyomegaly and intranuclear cytoplasmic invaginations.

While renal megalocytosis is sometimes seen in bovines that have succumbed to a severe chronic PA hepatopathy, it is usually only a minor change without clinically significant tubular injury.

In a study of bovine toxicosis (Shimshoni et al. 2015) where PA content of Heliotropum europium was analysed, histopathological examination of the livers revealed similar changes in all animals, with hyperplasia of bile ductules, prominent periportal fibrosis, cirrhosis with fibroblasts infiltrating the liver parenchyma and nodular regeneration. Megalocytosis was seen in the liver but not consistently seen in all samples. No pathological changes were observed in other organs including the kidneys.

Walker et al. 1981 reported focal or occasional megalocytosis of kidney tubular epithelium in cattle with hepatopathies due to natural and experimental Senecio lautus intoxication, although in one experimentally intoxicated calf a chronic focal non-suppurative interstitial nephritis around degenerating tubules was also noted.

In cases of bovine PA toxicoses submitted to Elizabeth Macarthur Agricultural Institute over the years, the major finding has been a chronic toxic hepatopathy. However, occasional megalocytosis of renal tubular epithelial cells was seen in a few animals, without other substantial damage to the kidney (EB personal experience).

The severe renal lesions in both these cows were similar, although appeared to be more chronic in the first cow (Case 1) necropsied, with extensive loss of tubules, leaving remnants of distorted tubules amongst prominent interstitial fibrosis. In the second cow (Case 10) most tubules remained in place, albeit severely damaged with degeneration, necrosis, loss of epithelial cells, collapse, pleiomorphism and megalocytosis, with only minimal replacement by fibrosis.

These renal lesions are unusual in bovines, and the authors are not aware of previous reports of severe nephropathy in cattle associated with ingestion of blue heliotrope, or other PA-containing plants.

Ketterer et al. (1987) reported on four cases of liver disease in cattle grazing H. amplexicaule in Southern Queensland. Histologically, livers of all cases had extensive hyperplasia of bile ducts and a variable degree of periportal and centrilobular fibrosis. Hepatocytes showed pronounced megalocytosis. Megalocytosis of renal cortical epithelium was evident only in two cases as a minor finding.

However, PAs, particularly those in Crotalaria sp, can target other organs than liver, particularly lung and kidney.

To the knowledge of the authors, amongst agricultural animals, only in pigs has the kidney been reported as the main target organ in PA toxicosis. In sheep with heliotrope toxicosis, renal changes are limited to those expected with secondary copper intoxication, with no mention of renal megalocytosis (Peterson et al. 1992).

Hooper et al. (1977) describe disease in pigs naturally and experimentally intoxicated with Crotalaria retusa. The dominant syndrome in the field outbreak was severe nephrosis and uraemia. A second syndrome was pulmonary disease. Both diseases could be co-existent or separate, and both were accompanied by microscopic pathological changes in the liver. All pigs with nephrosis had elevated urea nitrogen. Pulmonary disease was more dominant in experimentally intoxicated pigs while renal and hepatic lesions were present but did not kill the pigs. Microscopic lesions consisted of hepatic and renal tubular megalocytosis, and in those pigs developing nephrosis, tubular necrosis, tubular dilation, interstitial and periglomerular fibrosis, infiltration by monocytes and lymphocytes, protein casts, enlarged glomerular cell nuclei and glomerular atrophy were seen. Interstitial pneumonia was present in those with pulmonary disease.

Most of the features described in the kidneys of the pigs with nephrosis were also seen in the two cows that were necropsied. As in these cows, hepatic disease was a minor component in the pigs, largely limited to megalocytosis. In the feeding trial in the pigs, there was no clear relationship between the appearance of disease in an organ and the levels and duration of feeding of C. retusa.

Why these cows should display renal manifestations of PA toxicosis is open to speculation. There were no other known nephrotoxins including nephrotoxic plants in the paddock. An unknown factor may have enhanced the nephrotoxicity of H. amplexicaule. Temporary intraspecies differences like nutritional, metabolic and physiological status and underlying disease conditions could potentially alter the response to toxic insults and may influence the inclination of individual animals to eat the plants (Stegelmeier 2011).

Heliotrope is not very palatable to livestock and tends to be avoided, unless no other feed is available. However, some individuals continue to eat it indiscriminately (Agfact Common Heliotrope).

The amount of PAs in blue heliotrope is much higher than in common heliotrope (NSW WeedWise Blue Heliotrope). The types of PAs found in common heliotrope are considered more toxic than those found in blue heliotrope (Agfact Common Heliotrope).

Toxins are produced by the plants to deter herbivores (Jubb et al. 2016). The toxicity of a plant depends on many factors such as plant species, stage of growth, time of year and rainfall. Ketterer et al. (1987) mentions fertiliser application as another potential factor modulating toxic potential of blue heliotrope.

The potential for poisoning, diagnosis and prediction of poisoning, and metabolism of PAs and derivatives in the body are complicated. There are more than 350 potentially toxic PAs. Most plants have mixtures of these, all of which may be present in different forms with different toxic potential, and each one can be subject to different metabolic pathways. In the body they are metabolised by cellular (mostly hepatocellular) cytochrome P450 to their toxic derivatives, which are highly reactive and bind to many biomolecules, such as glutathione, facilitating excretion, but also cellular proteins and nucleic acids, damaging cell function. While hepatocytes are most affected, some produce extrahepatic damage in the lungs and kidney (Stegelmeier, Colegate et al. 2016).

In the two cows, the most common effect of PAs seen in ruminants, the antimitotic effect, was manifesting in a similar way even though considerably varying in degree in both liver and kidney. Antimitotic effects can be exerted via damage to microtubules. While DNA synthesis and mitosis are inhibited, some cells can replicate DNA without undergoing mitosis, resulting in megalocytes, with entrapped cytoplasm in some of the enlarged nuclei. Inhibited cells and megalocytes are long lived but eventually many undergo apoptosis (Jubb et al. 2016). These features of megalocytosis, intranuclear cytoplasmic invaginations and cell death, which usually are most prominent in the livers of intoxicated ruminants, were all present in the kidneys of these two cows, and together with the grazing history, were highly suggestive of an aetiological diagnosis of PA (blue heliotrope) nephropathy.

The factors determining the nephrotoxic potential of PAs and their derivatives in pigs and in the present bovine cases are uncertain. Metabolites of PAs are formed in any cells with adequate cytochrome P450 activity. Although the liver is the main site of bioactivation, other cells such as those in the proximal convoluted renal tubules can generate metabolites that cause local injury. Variation in the source of the toxin and in the species-based metabolic differences in the affected animals accounts for the differences in susceptibility of the different tissues (Jubb et al. 2016).

As the kidney is involved in removal of PA metabolites from the body one could speculate that, before or during this excretion process, something went wrong with detoxification and/or normally uneventful excretion of non-toxic forms in bovines, enabling toxic metabolites to exert similar effect on renal tubular epithelium as they do to hepatocytes. Another consideration is an impediment in the process of detoxification of PA compounds by rumen bacteria, a process that usually makes ruminants less susceptible to PA poisoning than monogastric animals (Loh et al. 2020). These cows had some evidence of concurrent problems, with the potential for marginal selenium status, parasitism, exposure to rock fern and state of pregnancy to contribute to disease.

Apart from classical liver disease, nephropathy as cause of death should be considered in ill thrifty, sick and dying cattle on pastures dominated by PA-containing plants. As in liver disease, disease may only manifest itself clinically with repeat or continuous exposure to the plants and may be exacerbated by additional stress factors. Should other cases arise further studies evaluating animal and environmental circumstances that bring on renal instead of hepatic disease would be of value.

Acknowledgements

We gratefully acknowledge the assistance of Andrew Orme, Identifications Technical Officer, from the Botanical Identification Service of the National Herbarium of NSW who identified Heliotropium amplexicaule.

References

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