Fifteen grazing beef cattle and calves presented a history of neurological signs like ataxia, intentional head tremors, muscle twitching. Nervous ketosis, nervous BVD, BHV-1,5, tremorgenic intoxication from hay, and Listeriosis were considered as differential diagnosis. Blood samples were collected. Inspection of hay bales showed large white dusty and moldy areas. Samples were taken and analyzed. Altered hay was immediately removed in all animals’ stock. No alterations were found in blood tests. Food analysis showed high concentrations of Roquefortine C (RC) (345 μg/kg DM). Tremorgenic syndrome has been reported in Penitrem A (PA) intoxication, but PA is difficult to isolate in laboratory conditions. Both RC and PA are produced by Penicillum spp. RC has been associated with PA in tremorgenic toxicosis in dogs and it might be considered a valuable diagnostic marker for PA intoxication. The neurological signs were due to tremorgenic intoxication after feeding of spoiled forage contaminated with mycotoxines

francesca bonelli

luca turini

micaela sgorbini

valentina meucci

English

Archivio della Ricerca - Università di Pisa

 1 Case Report 1 Using roquefortine C as a biomarker for penitrem A intoxication in a beef herd 2 F Bonelli1,2, V Meucci1, L Turini1,2*, M Sgorbini1,2 3 1Department of Veterinary Sciences, University of Pisa, Viale delle Piagge 2, 56124, Pisa, Italy. 4 2Centro di Ricerche Agro-Ambientali “E. Avanzi”, University of Pisa, via Vecchia di Marina 6, 56122, San Piero 5 a Grado (PI), Italy 6  7 *Corresponding Author 8 Dott. Luca Turini 9 Department of Veterinary Sciences 10 University of Pisa 11 Viale delle piagge 2, 56124, Pisa, Italy 12 Tel. +390502210115 13 Email: luca.turini@phd.unipi.it 14  15 State of novelty 16 • Incidence of Roquefortine C in livestock’ food is high reported, especially in visibly molded areas.  17 • Penitrem A is a well-recognized neurotoxic mycotoxin produced by Penicillium spp. which could induce 18 ataxia, tachypnea, and sustained tremors in dogs, cattle, sheep, rabbits, poultry and rodents. Penitrem A 19 is difficult to isolate in laboratory conditions. 20 • Roquefortine C has been associated with Penitrem A in tremorgenic toxicosis in dogs and it might be 21 considered a valuable diagnostic marker for Penitrem A intoxication. 22 • Roquefortine C has been found in forage of cattle affected by tremorgenic syndrome. 23  24   25  2 Abstract 26 Fifteen grazing beef cattle and calves presented a history of neurological signs like ataxia, intentional head 27 tremors, muscle twitching. Animals were fed with the same alimentary ration. Nervous ketosis, nervous BVD, 28 BHV-1,5, tremorgenic intoxication from hay, and Listeriosis were considered as differential diagnosis.  29 Blood samples were collected for CBC count, biochemistry panel, infectious diseases tests. Inspection of hay 30 bales showed large white dusty and moldy areas. Samples were taken and analyzed. Altered hay was immediately 31 removed in all animals’ stock.  32 No alterations were found in blood tests. Food analysis showed high concentrations of Roquefortine C (RC) (345 33 μg/kg DM). Tremorgenic syndrome has been reported in Penitrem A (PA) intoxication, but PA is difficult to 34 isolate in laboratory conditions. Both RC and PA are produced by Penicillum spp. RC has been associated with 35 PA in tremorgenic toxicosis in dogs and it might be considered a valuable diagnostic marker for PA intoxication. 36  37 Key words: bovine, mycotoxin, Penitrem A, Roquefortina C.   38  3 Introduction 39 Mycotoxins are low molecular weight compounds, metabolic products of the fungi growing on plants in the field, 40 or on forages during storage periods (Driehuis et al., 2008). Animal feed are naturally in contact with yeasts and 41 filamentous fungi present in the field, but contamination can also occur during harvesting, transport and storage. 42 The most common genera that are implied in ruminant mycotoxicosis are Aspergillus, Penicillium, Fusarium and 43 Alternaria (Keller et al., 2013). Exposure to mycotoxins through contaminated feed is one of the major risks 44 affecting ruminant health and toxic syndromes are different depending on the toxin involved (Keller et al., 2013). 45 Roquefortine C (RC) has been primarily associated with Penicillium roqueforti and Penicillium crustosum. 46 Presence of RC in livestock’ feed is highly reported, especially in silage because is both acid tolerant and able to 47 grow at low oxygen levels (Driehuis et al., 2008). RC intoxication causes anorexia, paralysis and ketosis in cattle. 48 High concentrations of RC seem also able to induce a shift in the rumen microflora composition (Driehuis et al., 49 2008). Despite has been showed that neurotoxic properties to RC (Shell, 2000), literature did not demonstrate 50 that RC could cause a tremorgenic syndrome. Penitrem A (PA) is a well-recognized neurotoxic mycotoxin 51 produced by Penicillium spp. which could induce ataxia, tachypnea, and sustained tremors in dogs, cattle, sheep, 52 rabbits, poultry and rodents (Shell, 2000; Tiwary et al., 2009). In mice, experimental intoxications with PA caused 53 seizures by increasing the release of glutamate, GABA and aspartate at cerebrocortical synapses and biochemical 54 reversible lesions (Barker et al., 2013). Diagnosis is based on clinical signs, demonstration of the mycotoxins 55 (RC and PA) in the feed, gastric fluid, urine, blood or feces (Barker et al., 2013). 56  57 History 58 The owner of an organic beef farm reported that several calves have been showed head tremors and stiffness gait 59 in the last two days (March 2016). Affected calves belonged to different “grazing groups” but were all kept in 60 neighbouring area. The owner did not report changes in the animals’ diet. 61 The herd was composed by a total 98 cows and their calves, kept in three different herds allocated by breed: 62 Limousine, Mucco Pisano and Chianina. A bull of each breed was also present, for a total of three bulls. The 63 animals were grazed all the year round in an area of 300 ha. According to the recommendations for organic 64 farming, hay, grass silage and maize silage were self-produced. Thus, the same daily ration was fed to all the 65 animals and the calves could eat the cows’ ration. Mineral supplementation did not take place. 66  4  67 Clinical Examination 68 The owner showed us 6 calves (between 1 to 5 months old, 4 males and 2 females) that presented neurological 69 signs. Three out of 6 calves have been left with their own dam, the other 3 presented more severe symptoms, thus 70 they were housed in a medication area. Due to the not cooperative attitude of the animals, the calves still kept in 71 their herds were only visually examined in the field; the neurological signs observed were ataxia, stiffness gait, 72 intentional head tremors and muscle twitching. We found 9 more affected animals: 6 calves (same age range, 5 73 males and 1 female) and 3 cows. The calves showed the same signs as described above, while the adults presented 74 mild ataxia. The remaining animals showed normal general body condition and attitude.  75 A complete physical examination has been performed on the 3 calves housed inside. All 3 calves showed 76 intentional head tremors and muscle twitching; 2/3 presented severe ataxia and stiffness gait, while 1/3 calf was 77 recumbent and unable to rise. The neurological examination showed deficits of the V and VII cranial nerves. 78 Calves could swallow, but they were unable to grab the food. The most important clinical data were: hyperthermia 79 (calf 1 – 39.9°C; 2 – 40.3°C; 3 – 40.8°C), tachypnea (calf 1 and 2 – 58 bpm; 3 – 72 bpm), tachycardia (calf 1 – 80 98 bpm; 2 – 104 bpm; 3 – over 120 bpm) and prolonged capillary refill time (CRT) (3-4’). Mild dehydration (6%) 81 was present in all the 3 calves (Fecteau, 2015). A slightly reduced motility of the rumen has been detected in all 82 the 3 animals. Both thorax sides presented normal auscultation and no nasal discharge was present. Joints were 83 all normal. 84 Totally, 15 animals were affected, 12/15 were calves between 1-5 months of age and 3/15 were cows. Calves 85 showed more severe signs than adults. One calf was euthanized due to poor general conditions, but necropsy was 86 not performed, according to the owner’s indication. 87 Macroscopic alterations of the silage, or the silos, could not be observed, while the hay presented deep alterations. 88 Diffuse and deep whitish areas were identified in some hay bales. This aspect became as much worse as the 89 inspection went deeper inside the bales. A total of 13 altered hay bales have been found in the storage area. The 90 owner reported that all the animals were eating hay coming from these bales and that he did not check the forage 91 before feeding the cows and calves. The inspection of the hay in the feeding area confirmed the presence of the 92 same alterations. 93  94  5 Diagnosis and Differential Diagnosis 95 The main differential diagnosis were tremorgenic mycotoxicosis, Listeriosis (Listeria monocytogenes), Bovine 96 Viral Diarrhea (BVD) nervous form, Bovine Herpesvirus type 1-5 (BHV1-5), and nervous ketosis. 97 Blood samples were collected for complete blood cell (CBC) count and biochemistry panel (total protein, urea, 98 creatinine, total and direct bilirubin, gamma-glutamyl transpeptidase, aspartate aminotransferase, creatine kinase, 99 and Magnesium). Urinalysis was performed for ketone bodies. Calves were also tested for infectious diseases 100 (Listeriosis, BVD, BHV 1-5). 101 Multiple samples from the contaminate hay were analyzed for mycotoxins and Listeriosis, but mycotoxins were 102 not evaluated in any biological samples. Mycotoxins were measured by using a validated HPLC/MS method. 103  104 Treatment adopted 105 CBC, biochemistry panel, and oligo-minerals resulted within normal ranges. All the animals were negative for 106 the infectious agents tested. No pharmacological treatments were made. 107 Feed analysis showed RC at level of 345 μg/kg dry matter - DM. Ready after sampling, all the hay in feeding 108 area and the contaminated hay bales were removed and destroyed. 109 All the animals, except the calf euthanized, recovered within 1 week. 110  111 Discussion 112 Initial clinical signs oriented to several differential diagnosis. Nervous ketosis was excluded because urinalysis 113 came negative for ketone bodies and calves are not considered a population at risk (Fleming, 2015). Cerebellar 114 hypoplasia caused by BVD virus induces intentional tremors and fever, but it is a condition typical of neonatal 115 or young calves and could not explain other nervous symptoms and the neurological signs in adults (George and 116 Metre, 2015). Tremors, fever and tachypnea might indicate BHV 1-5. However, besides increased respiratory 117 rate, which could be explained by the hyperthermia and muscle tremors, the animals did not show other 118 respiratory signs. The herd did not have an history of respiratory diseases, abortion or reproductive problems 119 (Smith et al., 2015). Intentional tremors, ataxia, fever, inability to grab the food and alterations of cranial nerves 120 might indicate Listeriosis. Also, outbreaks of listeriosis have been reported in late winter or early spring, 121 especially after periods of very heavy rain (Boileau and Gilliam, 2017). Some reports documented encephalitic 122  6 listeriosis as multiple cases over a short period of time. However, within a bovine herd, the prevalence of 123 encephalitic listeriosis is usually less than 5% (Boileau and Gilliam, 2017). The most common source of infection 124 for a cow is the silage, but encephalitic listeriosis has been observed in animals consuming pasture, hay and 125 soybean products (Boileau and Gilliam, 2017). Listeriosis was one of the most likely differential diagnosis in this 126 case. The owner refused to perform a punction of cerebro-spinal fluid, thus Listeria was only checked in the 127 forage and in the blood. Due to the macroscopically findings on hay, to the negative result concerning Listeriosis 128 and to the positive result about RC in the forage, a tremorgenic mycotoxicosis was considered the most likely 129 diagnosis and could explain both the more severe (intentional head tremors and muscle twitching, ataxia, stiffness 130 gait, recumbency, deficit of cranial nerves, hyperthermia, tachypnea and tachycardia), and the milder symptoms 131 (mild ataxia in adult cows) in animals fed with contaminated hay. Mild dehydration, prolonged CRT and rumen 132 hypomotility we found in few subjects were considered consequences of the main neurological signs. 133 Feed analysis performed was not able to find PA in detectable concentrations. In literature, it has been reported 134 that PA is more difficult to isolate than RC, but RC is always associated with PA in tremorgenic toxicosis caused 135 by PA (Tiwary et al., 2009). Thus, RC is considered a valuable diagnostic marker for PA intoxication (Tiwary et 136 al., 2009). Also, results for other potentially involved pathogens were negative. Therefore, a tremorgenic 137 mycotoxicosis due to PA-RC from Penicillium contamination was the final diagnosis.  138 No previous problems in storing had been detected. Unusual weather conditions might have created the right 139 circumstance for an extraordinary fungal grow and replication. 140 In conclusions, RC might be used in feed analysis and might represent a marker of PA allowing diagnosis of 141 tremorgenic intoxication in bovine. 142  143 Authors’ contribution 144 Bonelli, Turini and Sgorbini played a role in the clinical case evaluation and solving. Bonelli draft the manuscript, 145 while Turini and Sgorbini review it.  146 Meucci helped in the interpretation of results concerning Roquefortine C and also revised the manuscript. 147  148 References 149  7 1) Barker AK, Sthl C, Ensley SM, et al., 2013. Tremorgenic mycotoxicosis in dogs. Comp Cont Educ Pract Vet 150 E1-E5. 151 2) Boileau MJ and Gilliam J, 2017. Brainstem and cranial nerve disorders of ruminants. Vet Clin Food Anim 152 331(1): 67-79. 153 3) Driehuis F, Spanjer MC, Scholten JM, et al., 2008. Occurrence of mycotoxins in feedstuffs of dairy cows and 154 estimation of total dietary intakes. J Dairy Sci 91: 4261-71. 155 4) Fecteau ME, 2015. Fluid therapy in ruminants. In: Large Animal Internal Medicine (Smith BP, ed). 5th Ed, 156 Elsevier, USA, pp. 1387-90. 157 5) Fleming SA, 2015. Ketosis of ruminants. In: Large Animal Internal Medicine (Smith BP, ed). 5th Ed, Elsevier, 158 USA, pp.1252-8. 159 6) George LW and Van Metre DC, 2015. Cerebellar hypoplasia caused by congenital bovine viral diarrhea virus 160 infection. In: Large Animal Internal Medicine (Smith BP, ed). 5th Ed, Elsevier, USA, pp. 973-4. 161 7) Keller LAM, Gonzalez-Pereyra ML, Keller KM, et al., 2013. Fungal and mycotoxins contamination in corn 162 silage: monitoring risk before and after fermentation. J Stored Prod Res 52: 42-7. 163 8) Shell MM, 2000. Tremorgenic mycotoxin intoxication. Veterinary Medicine Publishing Group 1-2. 164 9) Smith OM, Van Metre DC and George LW, 2015. Encephalitis bovine herpesvirus infection. In: Large 165 Animal Internal Medicine (Smith BP, ed). 5th Ed, Elsevier, USA, pp. 938-9. 166 10) Tiwary AK, Puschner B and Poppenga RH, 2009. Using roquefortine C as biomarker for penitrem A 167 intoxication. J Vet Diagn Invest 21: 237-9. 168 

Using roquefortine C as a biomarker for penitrem A intoxication in a beef herd

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Using roquefortine C as a biomarker for penitrem A intoxication in a beef herd

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