Cattle are inherently susceptible to nitrate/nitrite poisoning due to their rumen’s metabolism of nitrate to the highly toxic intermediate product, nitrite1,7,8. This report describes a case of sudden death in Brahman cross vealers due to nitrate/nitrite toxicity as a result of feeding hay produced using effluent as a fertiliser.
A group of 130 juvenile mixed-sex Brahman cattle arrived in the early hours of 12 March 2020 at a property located near Casino, NSW, after a long journey from Queensland. The cattle were unloaded into a holding yard where they were supplied Rhodes grass (Chloris gayana) hay that had been produced on site and provided access to ad lib water. These animals were hungry after their travel and gorged on the hay supplied. No abnormalities in the behaviour or clinical appearance of the cattle were observed throughout the day. The following day, 13 March, it was discovered that 10 of the animals had died overnight.
The remaining 120 animals were separated from the 10 dead cattle. A veterinarian performed a physical examination on six of the surviving in-contact animals and reported no abnormalities. The veterinarian then contacted the local district veterinarian who discussed undertaking an anthrax exclusion and the possibility of nitrate poisoning. The veterinarian undertook an anthrax immunochromatographic test (ICT) pen-side test on blood-tinged nasal fluid from one of the dead animals—the result of which was negative. The veterinarian then sampled the aqueous humour from several of the dead animals, which revealed high nitrate levels on dipstick. At this stage the district veterinarian arrived at the property and a thorough post-mortem examination was performed on several of the dead animals.
The dead female animals had cyanotic udders, and all animals had brown/muddy coloured mucous membranes of the conjunctiva, oral cavity, and anus. Post-mortem examination was performed on two randomly selected dead animals. These animals had dark brown-coloured blood that was poorly clotted, petechial haemorrhages on serosal surfaces, and congestion of the liver, kidney, and lungs. Samples collected included aqueous humour, fresh and fixed liver, kidney, spleen, heart, and lung. Fresh and fixed samples of the brain were obtained from animal 1. Urine and abomasum content were collected from animal 1. Blood, rumen and intestinal content were obtained from animals 1 and 2.
Histopathology of animal 1 revealed mild multifocal myocardial haemorrhages present in the heart. Moderate amounts of protein-rich oedema fluid were present within alveolar lumina and interlobular septa of the lung. Mild lipid accumulation was present within hepatocytes, in addition to mild infiltration of lymphocytes and plasma cells within portal triads of the liver. No significant findings in the kidney, spleen, skeletal muscle, anterior brainstem, cerebral cortex, cerebellum or medulla were observed.
Histopathology of animal 2 revealed congestion of the lung with protein-rich fluid within alveolar lumina. Moderate numbers of large bacilli were present (post-mortem invaders), and the liver contained scattered areas of autolysis with large bacilli (post-mortem invaders) present.
The five samples of aqueous humour obtained by aqueocentesis were each examined on a nitrate/nitrite Merckoquant analytical strip test. The test results are presented below (Table 1). The samples were positive for nitrite, and the nitrate results were 250ppm in four animals and 50ppm in the other animal (normal concentrations are approximately 5ppm).
Table 1. Nitrate/nitrite Merckoquant Strip test results of aqueous humour.
|Sample ID||Sample Description||Nitrite||Nitrate (ppm)|
|NCMC 01||Occular fluid 01||+|
|NCMC 02||Occular fluid 02||+|
|NCMC 03||Occular fluid 03||+|
|NCMC 04||Occular fluid 04||+|
|NCMC 05||Occular fluid 05||+|
Displayed below in Table 2 is the DPI laboratory report for a hay sample submitted in November 2019. This sample of Rhodes grass hay was from the same batch cut from the same paddock as the Rhodes grass hay that was fed to these cattle upon arrival on 12 March 2020. This feed analysis was performed as mortalities on a separate property were suspected to be associated with the supplemental hay being fed. The hay producer at this time revealed that effluent had been used to fertilise the pasture, which was later cut to make hay. At the time it was advised that this hay should, ideally, not be used for livestock feed purposes.
Table 2. Feed analysis of a forage sample (Rhodes spp.) cut from the same paddock as the hay fed to these cattle in this case.
|Neutral Detergent Fibre (NIR)||%||10||69|
|Acid Detergent Fibre (NIR)||%||4||37|
|Water Soluble Carbohydrate (NIR)||%||4.0||4.4|
|Crude Protein (NIR)||%||2.0||11.5|
|Inorganic Ash (NIR)||%||3||9|
|Organic Matter (NIR)||%||75||91|
|AFIA Grade||No Grade|
|Metabolisable Energy (NIR)||MJ/kg DM||4.3||7.8|
Results below 3080mg/kg NO3, DM basis, are considered safe to feed if animals are on a normal ration. Concentrations above 9240 mg/kg NO3 is at a hazardous and potentially lethal concentration of intake for all animals. In this analysis the sample returned a level of 10397mg/kg, which is a dangerous level and therefore the sampled hay should only be fed with caution as part of a mixed ration to stock that are adapted and not hungry.
When plant material reaches the rumen non-protein nitrogen (NPN) and nitrate is converted to nitrite by rumen microflora4,8. Following this process, nitrite is converted to ammonia to be used by rumen microbes in producing microbial protein1,4. In excess, ammonia is absorbed across the rumen wall and metabolised by the liver7. Under circumstances where feed has an increased nitrate concentration this process becomes overwhelmed and nitrite accumulates to levels that exceed the capacity of rumen microbes to convert nitrate to ammonia,3,5. As a result, nitrite is absorbed into the animal’s blood stream, where it oxidises ferrous iron haemoglobin to ferric iron methaemoglobin5,7. Methaemoglobin is incapable of transporting oxygen, leading to tissue hypoxia2,8. Nitrite also affects the vasculature, causing vasodilation and hypotension, contributing to impaired oxygen delivery5. Hypoxia occurs when 20-30% of haemoglobin is converted to methaemoglobin, with death occurring after approximately 70-80% conversion7. Other effects of ingestion of high levels of nitrate include irritation of the gastrointestinal tract, which may lead to abdominal pain and diarrhoea5,7,8.
Risk factors for nitrate/nitrite poisoning include environmental and plant factors that lead to increased plant nitrate concentration, and animal factors such as stress and hunger6,8. Plants utilise nitrate from the soil to form amino acids via conversion of nitrate to ammonia. The conversion of nitrate to ammonia by the plant is a process that requires sunlight for energy, through photosynthesis4,6. Weather conditions with reduced sunlight, such as overcast weather, slow the rate of nitrate conversion. Furthermore, when plants become stressed during their growth stage nitrates can accumulate to toxic levels1,5,7. Plant stressors, including frost, drought, and insect damage, can all result in stunted growth where the conversion of nitrite to ammonia is reduced5,6. The excessive accumulation of nitrate in the plant during these periods poses a greater risk for toxicity in ruminants when grazing this forage.
Application of inorganic nitrogenous fertilisers or effluent to pastures is an inadvertent way that producers may cause forage to accumulate high levels of nitrate2. Furthermore, if there is a disruption to plant growth, as mentioned above, involving the pasture that has been fertilised then toxic accumulation of nitrate may be exacerbated5,7.
Allowing hungry animals to consume feed that has a high nitrate concentration puts them at a higher risk of toxicity as toxic events generally occur due to an acute intake of a high-risk forage over a short period1,6.
The clinical presentation of toxicity varies depending on the amount of nitrate consumed with higher amounts increasing the severity of disease. Acute toxic events may present clinically as dyspnoea, tachypnoea, tachycardia often with a weak heartbeat, hypothermia, ataxia, weakness, muscle tremors, diarrhoea, frequent urination, and the muddy brown, often cyanotic, mucous membranes4,5,7,8. Blood obtained from affected animals can have a mild to marked chocolate-brown appearance5,7. Petechial haemorrhages may be observed on serosal surfaces4,5. Recovered animals may have abortions or stillbirths if they have experienced approximately >50% methaemoglobin for six hours or longer4,7,8.
In this case, the first indication of disease was the finding of the 10 dead cattle. These cattle displayed no obvious signs of nitrate toxicity on the day that they were fed the hay. However, they may have shown clinical signs in the hours preceding death, which were not observed due to the cover of night. On the morning of the discovery, the remaining 120 cattle were determined to be clinically well by the attending veterinarian, who physically examined six randomly selected animals. However, the cyanotic udders of the deceased females, muddy-brown and cyanotic mucous membranes, and chocolate-brown blood in the rectum of the deceased animals are indicative of nitrate/nitrite poisoning2,4,6,7. Animals may die suddenly without developing clinical signs after exposure to highly toxic forage, or within a 24-hour period during which they may develop clinical signs relating to anoxia4,5,8.
The presence of blood in the nasal fluid indicated that anthrax should be ruled out as a differential for sudden death1,7. The attending veterinarian performed an anthrax immunochromatographic (ICT) exclusion test prior to conducting post-mortem examination, which returned a negative result.
The gross appearance of the deceased 10 animals was highly suggestive of nitrate/nitrite toxicity, so to investigate this possibility further the attending veterinarian performed aqueocentesis. This sample returned a positive result on Dipstick in the field, supporting this suspicion2. Analysis of the aqueous humour using a Merckoquant Strip test at the Elizabeth Macarthur Agricultural Institute (EMAI) was also positive, providing further support for the diagnosis. Post-mortem examination revealed congestion of the liver, kidney, and lungs, chocolate-brown coloured blood that was poorly clotted, and petechial haemorrhages on subcutaneous tissues, which are consistent with nitrate/nitrite toxicity2,3,4,6,7,8. Histological examination revealed changes that were not significant or diagnostic of any other differential2,3.
In this case a tentative diagnosis of nitrate toxicity was made.
A combination of risk factors contributed to the death of 10 out of 130 mixed-sex juvenile Brahman cattle animals due to nitrate/nitrite toxicity. The hay grown in drought conditions and fertilised with nitrogen-rich effluent contained dangerous levels of nitrates. The cattle were more susceptible to toxicity as they were hungry after being off feed during long-distance transport. Post-mortem findings and diagnostic testing supported this diagnosis.