Faecal thiaminase, plasma lactate and pyruvate concentrations and erythrocyte transketolase activity changes in apparently normal replacement ewes after the initiation to the pasture

A study was made to investigate faecal thiaminase and the thiamine-related biochemical changes in apparently normal replacement ewes with a feed change, after the initiation without adaptation to the new pasture. Twenty-four female ewes were divided into two groups. Group A was managed in a system based on pasture and was compared with group B system based on a diet of concentrate and straw until moving to pasture 9 weeks after. Blood samples for lactate, pyruvate and erythrocyte transketolase activity determinations and faeces for thiaminase estimation were evaluated chronologically. At the end of a 126 days experimental period, live weights of groups were similar. We confirmed that clinically normal sheep may have thiaminase activity in the faeces and concluded that the thiaminase release increased during the diet changes, from concentrate to pasture, and that their continued excretion could develop some degree of thiamine deficiency.     

Biochemical Changes in Apparently Normal Sheep from Flocks Affected by Polioencephalomalacia

Blood samples were obtained for determination of the activities of lactate, pyruvate and erythrocyte transketolase, and faeces as samples for estimation of thiaminase, from 190 apparently healthy sheep from 19 flocks in which at least one animal had been diagnosed with polioencephalomalacia (PEM), from 59 control animals and from 7 affected by PEM. Feed and pasture grass samples were collected for thiaminase analysis and thiaminase was assayed in ryegrass and fescue. Decreased erythrocyte transketolase activity, accompanied by a corresponding rise in the thiamin pyrophosphate effect, and estimation of the thiaminase content in faeces, may be useful diagnostic procedures by which to identify periods of greater risk, during which animals could develop PEM. The changes in the diet probably acted as a primary factor for microbial production of thiaminases in replacement ewes. In these, the prevalence was higher in the spring months.     

The effect of thiaminase-induced subclinical thiamine deficiency on growth of weaner sheep

Three experiments were performed to examine for causes of poor growth of young Merino sheep. Weekly testing of animals 42 weeks of age for 10 weeks revealed that 90% of clinically poor animals were excreting high levels of thiaminase in their faeces; low levels of activity were present in 20% of clinically normal animals. There were significant differences in the mean erythrocyte transketolase activity of the thiaminase excreting poor animals and the thiaminase free normal animals. Other known causes of poor growth could not be demonstrated. Weekly monitoring of thiaminase activity in the faeces from 80 lambs 6 weeks of age showed 23% to be excreting significant levels of enzyme (greater than 3mUg-1 DM) throughout a 10 week test period. Mean growth rates of these lambs were significantly below those of lambs not excreting thiaminase or excreting low levels intermittently. Supplementation of thiaminase excreting lambs with intra-muscular injections of thiamine HCl was associated with a statistically significant improved growth rate (P less than 0.01) compared to unsupplemented sheep excreting thiaminase. Mean growth rates of lambs not excreting thiaminase on a continuous basis (sampled weekly) were the same with or without thiamine HCl supplementation. High thiaminase levels were found in the ruminal fluids of trial animals excreting the enzyme in their faeces, confirming this previously established association. Bacillus thiaminolyticus was isolated from faeces and ruminal fluids from clinically poor animals and is the most likely source of the thiaminase. Subclinical thiamine deficiency was indicated by low erythrocyte transketolase activities and elevated TPP effects and is proposed as the cause of the poor growth by the young sheep.     

Thiamine deficiency in sheep exported live by sea

Three shipments of sheep being exported live by sea were examined to determine their thiamine status. Measurements were made of thiamine concentration in liver and ruminal contents, transketolase activity in erythrocytes and thiaminase activity in ruminal liquor. Sheep that died or were clinically ill and euthanased had significantly lower hepatic and ruminal thiamine concentrations than clinically healthy control sheep. A high proportion had thiamine concentrations comparable to those found in sheep that die with polioencephalomalacia. Thiamine concentrations decreased with increasing time that sheep were in pre-embarkation feedlots and on board ship. Destruction of thiamine in the rumen by thiaminase was not a significant factor. Erythrocyte transketolase activities indicated that many of the sheep that arrived in the Middle East without signs of clinical disease were also in a state of thiamine insufficiency.     

The effect of thiaminase-induced subclinical thiamine deficiency on growth of weaner sheep

Three experiments were performed to examine for causes of poor growth of young Merino sheep. Weekly testing of animals 42 weeks of age for 10 weeks revealed that 90% of clinically poor animals were excreting high levels of thiaminase in their faeces; low levels of activity were present in 20% of clinically normal animals. There were significant differences in the mean erythrocyte transketolase activity of the thiaminase excreting poor animals and the thiaminase free normal animals. Other known causes of poor growth could not be demonstrated. Weekly monitoring of thiaminase activity in the faeces from 80 lambs 6 weeks of age showed 23% to be excreting significant levels of enzyme (>3mUg^–1 DM) throughtut a 10 week test period. Mean growth rates of these lambs were significantly below those of lambs not excreting thiaminase or excreting low levels intermittently. Supplementation of thiaminase excreting lambs with intra-muscular injections of thiamine HCl was associated with a statistically significant improved growth rate (P<0.01) compared to unsupplemented sheep excreting thiaminase. Mean growth rates of lambs not excreting thiaminase on a continuous basis (sampled weekly) were the same with or without thiamine HCl supplementation. High thiaminase levels were found in the ruminal fluids of trial animals excreting the enzyme in their faeces, confirming this previously established association.Bacillus thiaminolyticus was isolated from faeces and ruminal fluids from clinically poor animals and is the most likely source of the thiaminase. Subclinical thiamine deficiency was indicated by low erythrocyte transketolase activities and elevated TPP effects and is proposed as the cause of the poor growth by the young sheep.     

Oral treatment of polioencephalomalacia and subclinical thiamine deficiency with thiamine propyl disulphide and thiamine hydrochloride

Thiaminase type I production by Bacillus thiaminolyticus and activity in vitro were repressed by the primary substate thiamine and by thiamine monophosphate and thiamine propyl disulphide. At thiamine concentrations of 300-3000 mumol/l production of active enzyme by B. thiaminolyticus, and activity of purified enzyme, were totally repressed. Growth of B. thiaminolyticus was inhibited by thiamine propyl disulphide at 3000 mumol/l. Activity of purified thiaminase was lost when incubated with ruminal fluid from healthy thiaminase free sheep. Enzyme activity was also lost when exposed to mildly alkaline conditions but was stimulated and stabilized against heat denaturation if treated with dithiothreitol. Thiaminase activities in the ruminal fluids of cases of ovine polioencephalomalacia were repressed within 2 h of oral administration of thiamine propyl disulphide. Blood pyruvate levels and transketolase activities were restored to normal following treatment. Treated animals recovered clinically and were returned to pasture.     

Induction of bovine polioencephalomalacia with a feeding system based on molasses and urea.

Polioencephalomalacia (PEM), a disease first described in the United States and related to intensive beef production, appeared in Cuba coincident with the use of a new, molasses-urea-based diet to fatten bulls. Because the only experimental means so far of reproducing PEM has been with amprolium, a structural analog of thiamin, the present study attempted to induce the disease using the molasses-urea-based diet. Six Holstein bulls (200-300 kg) were studied during consumption of three successive diets: 1) commercial molasses-urea-restricted forage diet of Cuban feedlots, 2) a period in which forage was gradually withdrawn and 3) a forage-free diet composed only of molasses, urea and fish meal. PEM was reproduced in this way. At ten-day intervals, blood concentrations of glucose, lactate, pyruvate and urea were measured, as well as when clinical signs of PEM appeared. The signs, clinical course and lesions of the experimentally induced disease were comparable to those of field cases. The biochemical results suggested a block in pyruvate oxidation as in PEM elsewhere in the world. No evidence existed of urea intoxication. In addition, brain and liver concentration of total thiamin from field cases and normal animals were found to be similar.     

Sulfur-induced polioencephalomalacia in sheep: some biochemical changes.

The effect of high dietary sulfur (S) supplementation on blood thiamine (B1) concentration, biochemical indices of liver, muscle and kidney damage and selected plasma electrolytes was studied in six sheep. Three of these sheep received an additional 230 mg thiamine/kg diet (Group 2). After approximately 2.5-3 weeks on this diet, all three sheep in the non-B1-supplemented group (Group 1) showed loss of appetite and developed mild neurological signs: depression, intermittent signs of excitation and head pressing. Increases in blood B1 concentration and plasma creatine kinase (CK) and aspartate aminotransferase (AST) were observed during this time in all affected animals. Clinical signs lasted only for two to five days. Sheep in group 2 were clinically normal throughout the experiment, but all of these animals also had elevated blood B1 concentrations and plasma CK activity at the 3 wk sampling. Plasma magnesium concentrations of group 1 sheep were elevated at the 2.5-3 wk and 6 wk samplings but they declined significantly (p less than 0.05) to low normal levels thereafter. Magnesium concentrations of group 2 sheep were low at the beginning but progressively increased during the course of the experiment. At necropsy, brain lesions suggestive of polioencephalomalacia (PEM) were observed in all sheep but were most marked in group 1. It is speculated that PEM may be caused by a direct toxic effect of S, S metabolites or B1 antimetabolites in the brain rather than by an in vivo B1 deficiency per se.     

Natural establishment of thiaminase activity in the alimentary tract of newborn lambs and effects on thiamine status and growth rates

Thiaminase activity was detected in the faeces of lambs at 2 to 5 days of age. Levels of activity increased for 10 days and then declined over the next 3 to 4 weeks. Decreased erythrocyte transketolase activity indicated thiamine insufficiency in lambs with high thiaminase activity. Mean growth rates were 17% less in lambs with high thiaminase activity than in lambs with zero or low thiaminase activity. Bacillus thiaminolyticus was the only organism isolated which produced thiaminase. Treatment of newborn lambs with intramuscular injections of sulphadoxine did not prevent them from excreting thiaminase in their faeces. It is proposed that oral thiamine supplementation of lambs at 2 to 3 weeks of age may be the most appropriate prevention and treatment for subclinical thiamine deficiency of the cause described.     

Role of thiamine status in sulphur induced polioencephalomalacia in sheep.

The effects of excess dietary sulphur were studied in sheep supplemented and unsupplemented with thiamine. The diets contained either 0.19 per cent sulphur (LS) or 0.63 per cent sulphur (HS) in combinations with 14 mg kg-1 thiamine (LB1) or 243 mg kg-1 thiamine (HB1). A total of 56 two-month-old lambs were used. Groups consisting of nine, nine, 22 and 16 lambs were fed LS-LB1, LS-HB1, HS-LB1 and HS-HB1 diets, respectively for 14 weeks. Out of 22 lambs fed the HS-LB1 diet, seven lambs developed neurological signs between the third and eighth week of the trial. Two of these lambs died, three that were in extremis were euthanased, and two recovered completely. All clinically affected animals had extensive malacic lesions in the cerebral cortex, midbrain and brainstem. None of the lambs from the LS groups or HS-HB1 group developed clinical signs. Several clinically normal lambs from the HS-LB1 group had necrotic lesions in their brains at gross and microscopic examination. Supplementation with dietary thiamine prevented development of clinical signs, but did not totally prevent development of microscopic brain lesions. Brain thiamine concentration, transketolase activity and thiamine pyrophosphate (TPP) effect were not different (P greater than 0.05) among groups. There was a strong effect (P less than 0.0001) of dietary thiamine supplementation on blood thiamine concentration and TPP effect. Blood thiamine concentration was higher whereas TPP effect was lower in the thiamine supplemented sheep. Blood and tissue thiamine concentrations in sheep exposed to high dietary sulphur did not indicate either systemic or local thiamine deficiency per se. Increased TPP effect in sheep fed the HS-LB1 diet indicated mild to moderate metabolic thiamine deficiency. Thiamine inadequacy may be an effect of an increased requirement for thiamine in animals exposed to excess dietary sulphur.     

Pyrimidinyl nicotinic acid and cerebrocortical necrosis.

Pyrimidinyl nicotinic acid (PNA) exhibits great structural similarities to amprolium, which is a known thiamine antagonist. Experimental cerebrocortical necrosis (CCN) has been induced by oral administration of amprolium. PNA has been demonstrated in ruminai fluid from animals suffering from CCN.The aim of this investigation was to study whether PNA can act as thiamine antagonist and whether it can give rise to CCN. For this purpose PNA was synthesized and was daily given intravenously to a calf. The dose corresponded to roughly 10 times the content of thiamine in the blood. After three weeks the dose was doubled. During the entire experimental period comprising nine weeks no clinical sign of thiamine deficiency or CCN was noticeable. The values for all recorded blood chemical parameters, with the exception of occasional GOT and PK values, were within the normal limits of variation.Rats were used in a similar experiment with the same aim. PNA was homogeneously added to their feed in quantities equivalent to five and 10 times the thiamine content. The rats were clinically healthy throughout the experimental period comprising eight weeks. No significant difference in TK activity and TPP effect was observed between the experimental groups and the control group.     

Electroencephalographic observation on sheep and cattle with experimental cerebrocortical necrosis

Nine Suffolk sheep and 4 Holstein cattle were employed to observe electroencephalographic changes in experimental cerebrocortical necrosis. Amprolium (600 mg/kg/day) alone was given intraruminally 6 sheep and 4 cattle, all of which showed neurological signs and abnormal electroencephalograms. Both amprolium and thiamine (200 mg/day) and thiamine (50 mg/day) alone were given 2 and 1 sheep, respectively, as controls, which did not show any abnormal signs. Abnormal electroencephalograms included continuous slow waves and long-lasting spindles, both of which appeared diffusely and were seen subclinically or with neurological signs. The spindles consisted of slow waves or sharp- (or spike-) and-slow-wave complexes with or without convulsive seizures even during the convulsion stage. Conversely, the seizures occurred with the spindles or slow waves.     

Clinical and biochemical alterations in calves with nutritionally induced polioencephalomalacia

Polioencephalomalacia (PEM) was induced in calves by feeding a semipurified, low-roughage diet of variable copper and molybdenum composition. Two formulations resulting in Cu-insufficient and Cu-sufficient forms of the diet were fed (n = 10 and 4 calves, respectively); both diets induced PEM. Clinical signs of disease developed as early as 15 days after transition to the experimental diets and included impaired vision, decreased response to external stimuli, and abnormal gait. Grossly evident cerebrocortical lesions consisted of laminar areas of cavitation and/or autofluorescence seen under UV illumination. Hepatic Cu concentration was decreased in calves fed the Cu-insufficient diet, but not below normal range. During the course of feeding either diet, rumen pH decreased, rumen volatile fatty acid concentrations increased, rumen and blood lactic acid concentrations increased, and rumen and plasma thiamine concentrations increased. The thiamine pyrophosphate effect on erythrocyte transketolase activity was unaltered in calves of either diet group. This nutritionally induced form of PEM does not appear to be related to Cu deficiency or reduction in plasma or rumen thiamine concentration.     

Clinical and biochemical findings in bovine cerebrocortical necrosis produced by oral administration of amprolium

Purposing to get some hints on cause and early diagnosis for cerebrocortical necrosis (CCN), CCN was produced in three healthy calves by the oral administration of amprolium. All three calves showed central nervous signs characterized by ataxic gait, clonic spasm, astasia and opisthotonus, from 24 to 49 days after the start of daily administration of 321-418 mg/kg amprolium. They showed bradycardia from about 20 days before the appearance of the nervous signs, which was supposed to be a finding of primary change and to be useful for early diagnosis of CCN. At necropsy of the two calves, large necrotic lesion was found in the cerebral cortex, and tissue thiamine levels decreased significantly, especially in cerebrum and cerebellum. In the other calf, injection with 25 mg thiamine tetrahydrofurfuryl disulfide (TTFD) was proved to be effective for the recovery of clinical signs. No significant changes in thiamine level were recorded in the whole blood, but those in erythrocytes decreased slightly at about a week before the appearance of the clinical signs. No significant alteration of thiamine excretion was observed in urine. Those findings suggest that CCN in calves is caused by thiamine deficiency and that the blood thiamine levels cannot be used for diagnosis of CCN.     

Polioencephalomalacia in adult sheep grazing pastures with prostrate pigweed

Polioencephalomalacia was diagnosed in 2 animals from different farms. In apparently healthy animals from same farms, fecal thiaminase and a significant reduction in erythrocyte trans-ketolase activity was observed. The presence of thiaminase in Amaranthus blitoides could have contributed to the development of polioencephalomalacia in sheep grazing on natural pastures.     

Severe thiamine deficiency in sheep with acute ruminal lactic acidosis

BACKGROUND: Thiamine status of ruminants is adversely affected in acidic rumen conditions. However, there have been limited published case study data related to thiamine deficiency of ruminants with acute ruminal lactic acidosis (ARLA). HYPOTHESIS: Thiamine deficiency would occur in sheep with ARLA. ANIMALS: Thirteen Ak-Karaman (white Karaman) sheep with ARLA, aged 1 year (ARLA group) and 10 healthy Ak-Karaman sheep, aged 1 year (control group) were used. METHODS: After clinical examination, rumen fluid samples of all sheep were obtained with a stomach tube and examined immediately. Blood samples were taken from a jugular vein of the sheep. Erythrocytic transketolase enzyme activity and hence thiamine pyrophosphate (TPP) effect were determined according to Clausen's method. RESULTS: History revealed that all sheep in the ARLA group had accidentally consumed excessive amounts of cracked barley. During clinical examination of the ARLA group, disturbed general condition, engorged scleral vessels, moderate to severe dehydration, and ruminal atony were recorded in the sheep. The results of the ruminal fluid analyses of the ARLA group demonstrated characteristics of ARLA. The results of clinical and ruminal fluid examination of control group were normal. The mean TPP effect (%) in the ARLA group (109 +/- 28) was significantly higher than in the control group (22.2 +/- 3.7) (P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: The present study revealed that severe thiamine deficiency occurred in sheep with ARLA. This result indicates that thiamine administration to sheep suffering from acute ruminal acidosis caused by overconsumption of readily fermentable carbohydrates could be beneficial in alleviating thiamine deficiency caused by ruminal acidosis.     

Cerebro-cortical necrosis (C.C.N.) in calf. An experimental reproduction of the disease

In four ruminant calves a state of thiamine deficiency was brought about with a thiamine antagonist, amprolium. Twice daily the calves were given Amprolmix® containing 25 % amprolium through a stomach tube. The dosage was 350–500 mg/kg body weight daily during the entire experimental period. After 31 days the first calf showed fully developed symptoms; two other calves fell ill after another four and eight days respectively. The fourth calf was killed before the disease became clinically manifest. In all calves cerebrocortical necroses were found on histological examination. The clinical picture is described. It was similar to that in spontaneous C.C.N.In the calves exhibiting fully developed symptoms a moderate rise was observed in the pyruvate kinase and a heavy rise in the creatine Phosphokinase activity. On several occasions the total quantity of thiamine (free + phosphorylated) in the blood exceeded the upper normal limit without any extra supply of thiamine being given. Only on one occasion was a lowering observed, to a value just below the lower limit of normal variation.     

Ruminant thiamine requirement in perspective

Thiaminases play an important role in the aetiology of CCN being responsible for the state of thiamine-deficiency which is an essential feature of the disease, evidence for which is presented here. These studies have led to a greater appreciation of the role of thiamine and thiaminases in ruminant nutrition especially as ruminal thiaminase activity is not confined to clinically affected animals but is of wider distribution. The importance of thiaminases in intensive beef production and the possibility of the need for thiamine supplementation in the form of a thiaminase resistant derivative is discussed.     

Clupeid Response to Stressors: The Influence of Environmental Factors on Thiaminase Expression

Abstract

Over the past five decades, a reproductive failure related to thiamine deficiency, referred to as thiamine deficiency complex (TDC), has been observed in valuable salmonine fishes in the Great Lakes and Finger Lakes in North America and the Baltic Sea in Europe. The cause of TDC has been linked to the consumption of clupeid fish, which contain high levels of a thiamine-destroying enzyme called thiaminase I (hereafter referred to as “thiaminase”). High activities of thiaminase have been reported from clupeids such as Alewife Alosa pseudoharengus, Gizzard Shad Dorosoma cepedianum and Atlantic (Baltic) Herring Clupea harengus, but no consistent explanation has accounted for the wide range of observed variation in levels of thiaminase in clupeids. Chronic stress can suppress the immune systems of Alewife and other fishes, thereby reducing the number of circulating white blood cells available to suppress bacteria. Because the presence of thiaminase has been associated with thiaminolytic bacteria isolated from Alewife viscera, we hypothesized that stressful conditions, which can potentially limit clupeid immune response or alter internal physiological conditions, could allow for thiaminase to be produced more efficiently by bacteria or thiaminolytic bacteria could proliferate, or both events could occur, resulting in a subsequent increase in thiaminolytic activity. In this study, Alewives and Gizzard Shad were exposed to severe winter temperatures and low food availability, respectively, in replicated pond experiments to evaluate the influence of stressful conditions on clupeid thiaminase activity. Though responses in circulating white blood cell counts and metrics of fish condition indicated that experimental treatments affected these clupeids, these effects were not related to increased thiaminase activity. The only significant treatment effect on clupeid thiaminase was an increase in mean thiaminase activity in Gizzard Shad from ponds where only high quality energy sources were available. These data indicate that variability in clupeid thiaminase may be related to diet composition.

Received June 19, 2012; accepted January 15, 2013     

Polioencephalomalacia in cattle: a consequence of prolonged feeding barley malt sprouts

Polioencephalomalacia (PEM) in ruminants has been recognized as a consequence of excess sulphur intake. The present study describes clinical, gross and histopathological findings of PEM following an abrupt change of diet in two ranches housing 2750 dairy and 2300 beef cattle. As a result of severe PEM, 256 cattle died or were slaughtered. Clinical findings included circling, hypersensitivity, excessive salivation, hypermetria, incoordination, blindness and death. The first clinical signs occurred in beef calves (6-8 months old) at a holding facility. Clinical signs of the disorder continued intermittently during the 5-month period in both ranches and were more evident in calves and lactating dairy cows. The affected cattle did not respond to thiamine injections. Clinical signs disappeared gradually following removal of barley malt sprouts from the diet. Although macroscopic lesions were not apparent in the brain tissues of some animals, histopathology typical of PEM was found in most cases: spongiosis in the neuropil and neuronal necrosis, haemorrhage, capillary hyperplasia, fibrinoid degeneration in arterioles, multifocal liquefaction necroses in the grey matter and abundance of gitter cells with vacuolar large cytoplasm. Sulphide in rumen fluid of a clinically affected animal was measured as 1.55 mg/dl, which is considerably higher than that collected from two control cows (mean 0.21 mg/dl). The total sulphur content of the diet containing barley malt sprouts was estimated to be 0.45%, which is also higher than the National Research Council (NRC) maximum tolerable levels. In conclusion, PEM can result from excess barley malt sprout intake because of its higher sulphur content. Clinical signs may occur shortly after the intake of barley malt sprout as outbreaks with a higher number of deaths or as an ongoing periodic condition.