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.     

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.     

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.     

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.     

Development of Thiamine Deficiencies and Early Mortality Syndrome in Lake Trout by Feeding Experimental and Feral Fish Diets Containing Thiaminase

Abstract

We conducted a laboratory investigation on the consequences of feeding predatory salmonids either experimental diets low in thiamine or diets containing alewife Alosa pseudoharengus. In experiment 1, adult lake trout Salvelinus namaycush were fed experimental diets containing bacterial thiaminase. In experiment 2, adult lake trout were fed natural prey species, alewives, and bloaters Coregonus hoyi. The diets consisted of four combinations of alewives and bloaters from Lake Michigan (100% alewives, 65% alewives–35% bloaters, 35% alewives–65% bloaters, and 100% bloaters), alewives from Cayuga Lake, a casein bacterial thiaminase, and a commercial trout diet. We assessed the effects of each diet on egg thiamine concentration and incidence of an embryonic early mortality syndrome (EMS). In experiment 1, incidence of EMS ranged from 0% to 100%. Significant relationships were found between the incidence of EMS and thiamine. In experiment 2, adult lake trout fed 100% alewives from either Lake Michigan or Cayuga Lake or fish fed the casein bacterial thiaminase diet produced eggs with low thiamine and swim-up fry with EMS. At either 35% or 65% alewives in the diet, egg thiamine was significantly lowered. The number of females that produced offspring that died from EMS were low but demonstrated the negative potential if feral lake trout foraged on either 35% or 65% alewives. Depleted egg thiamine and the onset of EMS required diets containing thiaminase for a minimum of 2 years in lake trout initially fully thiamine replete. We conclude that EMS can be caused by extensive feeding on 100% alewives and dietary levels of 35% or greater may prove detrimental to sustainable reproduction of salmonids in the Great Lakes. The data are consistent with that observed in feral lake trout, and it is concluded that EMS is the result of a thiamine deficiency.     

Thiaminase Activity of Crucian Carp Carassius carassius Injected with a Bacterial Fish Pathogen,Aeromonas salmonicida subsp. salmonicida

Abstract

Dietary thiaminase I is a cause of thiamine deficiency in animals. The physiological significance of thiaminase in the organisms containing this enzyme is not known, nor are the factors causing variation in their thiaminase activity. Tests were performed to evaluate the effect a pathogen might have on thiaminase activity in fish, when analyzed both with a cosubstrate added (CATA tests) and no cosubstrate added (NCATA tests). Pyridine is known as a cosubstrate specific for thiaminase I activity that does not accelerate thiaminase II activity. Crucian carp Carassius carassius known to harbor thiaminase I activity were injected intramuscularly with live Aeromonas salmonicida, a pathogenic bacterium of fish. For comparison, other groups were injected with formalin-killed bacteria and phosphate-buffered saline, respectively; an untreated group of fish was kept as a control. The bacteria did not contain any thiaminase activity. Significantly higher thiaminase activities (CATA and NCATA) were measured in all tissues (whole blood, injected muscle, uninjected muscle, and whole fish homogenates) of fish injected with live bacteria than in the saline-injected and the uninjected groups. The thiaminase activity of blood and that in the injected, inflamed muscle tissue followed different allocation patterns in fish injected with live A. salmonicida. The amount of thiaminase I enzyme appeared to be elevated in the whole blood of injected fish in the absence of natural cosubstrate(s). The thiaminase activity of the injected, inflamed muscle suggested that both the amount of thiaminase enzyme and some yet-unidentified natural cosubstrate(s) were elevated. This suggests that in addition to the enzyme, some cosubstrate(s) of fish or pathogen origin play a regulatory role in the so-far-unknown physiological significance of thiaminase I activity in vivo. It is suggested that the health of fish should be considered when searching for factor(s) affecting its thiaminase activity.     

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     

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.     

BIOCHEMISTRY OF NATURAL AND AMPROLIUM-INDUCED POLIOENCEPHALOMALACIA IN SHEEP

SUMMARY Polioencephalomalacia (PEM) induced in sheep was compared with the disease found in naturally occurring cases. Blood biochemical indicators measured were pyruvate, lactate, glucose, erythrocyte transketolase (TK) and stimulation of TK by addition of thiamine pyrophosphate (TPP effect). Faeces and rumen contents were assayed for thiaminase activity. The effect of treating affected sheep with thiamine was also noted. It was found that amprolium treatment could induce thrombocytopenia, but once the sheep became accustomed to amprolium in the diet they seemed to be resistant to this effect. In sheep receiving amprolium significant weight losses preceded the onset of clinical signs. Further weight loss continued throughout the recovery period despite removal of amprolium from the diet and treatment with thiamine. Blood glucose was variable, and was elevated only when marked clinical signs were present. Pyruvate and lactate levels showed marked variation throughout the trial. TK values were depressed and TPP effects increased well before the onset of clinical signs, although some naturally occurring cases had normal levels. Faecal thiaminase activity was negligible in all the sheep on the amprolium trial but most field cases had a high level. High faecal thiaminase was observed in about 5% of clinically normal animals from affected flocks. Depression of erythrocyte TK activity coupled with the presence of faecal thiaminase appeared to be the most reliable diagnostic biochemical parameters for PEM. Treatment of PEM affected sheep with thiamine rapidly brought the biochemical status of the animals to normal. However where advanced brain lesions were present the damage was permanent and such sheep treated with thiamine remained partially decorticate.     

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.     

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.     

Thiaminase activities and thiamine content of Pteridium aquilinum, Equisetum ramosissimum, Malva parviflora, Pennisetum clandestinum and Medicago sativa

Thiaminase type 1 and 2 activities and thiamine content of five plants were determined. Of these Pteridium aquilinum and Equisetum ramosissimum were found to have considerably more thiaminase activity and lower thiamine content than Malva parviflora, Pennisetum clandestinum and Medicago sativa.     

Quantitative analysis of thiaminase activity in certain fish species.

Thiaminase I and II activity of Baltic herring, vendace, smelt and dace was measured. All four fish species were found to contain thiaminase activity. The amounts of thiaminase activity in mug of thiamine split per 100 g of fish tissue per hour at 37 degrees C were: in Baltic herring 115 +/- 60, in dace 11500 +/- 2050, in smelt 25 +/- 25 and in vendace 30+/- 15.     

Implications of Thiamine Deficiency in Great Lakes Salmonines

Abstract

Our recent experimental work and ecoepizootiological assessments provide mechanistic data supporting a plausible hypothesis for an association between a prey base comprised of a large biomass of nonnative alewives Alosa pseudoharengus and the recruitment difficulties currently experienced by Great Lakes salmonines. We hypothesize that the thiamine deficiency induced by alewives, a species harboring high thiaminase activity, represents an ongoing cause of fry and adult mortality in salmonines. Overall ramifications of the thiamine deficiency on recruitment have not been firmly established but may represent a substantial bottleneck for natural recruitment in feral salmonine populations in the Great Lakes.     

Effectiveness and retention of thiamine and its analogs administered to steelhead and landlocked Atlantic salmon

We investigated the feasibility of enhancing the reproduction of steelhead Oncorhynchus mykiss and landlocked Atlantic salmon Salmo salar in lakes where the consumption of alewives Alosa pseudoharengus and other forage fishes containing thiaminase can cause them to become thiamine deficient and thereby reduce the survival of their fry. We evaluated feeding fingerling steelhead excess thiamine hydrochloride (THC1) for 1 or 2 weeks or equimolar amounts of thiamine mononitrate, thiamine-tetrahydrofurfuryl-disulfide, benfotiamine, or dibenzoyl thiamine (DBT). We found minimal internal reserves of thiamine after 6 months. We also compared the ability of injections of thiamine and its analogs to prevent mortality in thiamine-deficient steelhead and Atlantic salmon sac fry and found all forms to be effective, although benfotiamine was the least effective on an equimolar basis. Further, we injected yearling steelhead and found that DBT was tolerated at approximately 11,200 nmol/g of body weight, about 10 times more than thiamine in any other form. When yearling steelhead were injected with near-maximal doses of thiamine hydrochloride and several analogs and then fed a thiamine-deficient diet, DBT was retained for approximately 2 years--in contrast to other forms, which were retained for less than about 6 months. Therefore, these results suggest that neither feeding nor injecting young hatchery salmonids with DBT is likely to enhance their reproduction for more than 2 years after stocking. However, injecting DBT in nearly mature fish (either cultured fish from hatcheries or wild fish captured in lakes) may provide them with enough thiamine to successfully spawn within 2 years even though they consume mainly thiaminase-containing forage fishes.     

Thiamine and fatty acid content of walleye tissue from three southern U.S. reservoirs

We determined the thiamine concentration in egg, muscle, and liver tissues of walleyes Sander vitreus and the fatty acid content of walleye eggs from three southern U.S. reservoirs. In two Tennessee reservoirs (Dale Hollow and Center Hill), in which there were alewives Alosa pseudoharengus in the forage base, natural recruitment of walleyes was not occurring; by contrast in Lake James Reservoir, North Carolina, where there were no alewives, the walleye population was sustained via natural recruitment. Female walleye tissues were collected and assayed for thiamine (vitamin B1) and fatty acid content. Thiamine pyrophosphate was found to be the predominant form of thiamine in walleye eggs. In 2000, mean total egg thiamine concentrations were similar among Center Hill, Dale Hollow, and Lake James reservoirs (2.13, 3.14, and 2.77 nmol thiamine/g, respectively). Egg thiamine concentration increased as maternal muscle (r2 = 0.73) and liver (r2 = 0.68) thiamine concentration increased. Walleye egg thiamine does not appear to be connected to poor natural reproduction in Tennessee walleyes. Threadfin shad Dorosoma petenense, which are found in all three reservoirs, had higher thiaminase activity than alewives. Six fatty acids differed among the walleye eggs for the three reservoirs. Two were physiologically important fatty acids, arachidonic acid (20:4[n-6]) and docosahexaenoic acid (22:6[n-3]), which are important eicosanoid precursors involved in the regulation of biological functions, such as immune response and reproduction.     

Evaluating the effect of stressors on thiaminase activity in alewife

No consistent explanation has been found for the variability in the thiaminase activity of alewives Alosa pseudoharengus despite the role of alewife thiaminase in large-scale salmonine mortality in the Laurentian Great Lakes. We conducted experiments to evaluate the effect of two stressors, reduced salt content in the water and food limitation, on alewife thiaminase activity. Alewives were subjected to treatments in replicated tanks in which conductivity was lowered (< 100 microS/cm) for 8 d and feeding was limited for 39 d. Circulating white blood cells, plasma cortisol, plasma glucose, and whole-body thiaminase were measured in individual alewives to assess their response to these experimental treatments. Alewives from the controls had significantly larger numbers of circulating white blood cells than those in the salt-reduced and food-limited treatments (24,000 and 19,000 cells/microL and 11,000 and 9,000 cells/microL for alewives from the two control and salt-reduced treatment tanks, respectively, and 34,000 and 30,000 cells/microL and 21,000 and 16,000 cells/microL for alewives from the two control and food-limited treatment tanks). No significant differences in alewife thiaminase activity were found between treatment fish and their controls. The mean thiaminase activity in the alewives studied increased from 6,900 to 16,000 pmol x g(-1) x min(-1) from the time of their collection in Cayuga Lake to the start of laboratory experiments 1.5-2.5 years later; the latter value was more than twice that of previously reported levels of thiaminase activity from alewives collected in the wild. These data suggest that the variability in alewife thiaminase is not related to stress from salt reduction or food limitation, but laboratory holding conditions significantly increased thiaminase through a mechanism not evaluated by our experimental treatments.     

Thiamin and niacin in the rumen

Thiamin analogs, produced in the rumen by thiaminase I, in the presence of a cosubstrate appear to be responsible for the central nervous system disorder, polioencephalomalacia (PEM). For PEM to occur, an analog must be produced that inhibits an essential thiamin-requiring reaction, and results from a cosubstrate present in the rumen. In high concentrate diets, thiaminase I is produced by rumen microbes. However, PEM can also be caused by thiaminase I of plant origin. Based on physical characteristics and cosubstrate specificity, the thiaminase I enzymes produced by Bacillus thiaminolyticus and Clostridium sporogenes appear to be different from the enzyme produced by the rumen. Because niacin and certain antihelmentics are thiaminase I cosubstrates, they should be used cautiously. Supplementary niacin increased microbial protein synthesis in vitro and in vivo, and was more effective with urea than soybean meal. Supplementary niacin (5 to 6 g X cow-1 X d-1) increased milk production in postpartum cows but not in those in mid-lactation, and in cows fed soybean meal but not in those fed urea. We believe the heating of soybean meal during commercial processing decreased the availability of niacin for rumen protozoa. Supplementary niacin for postpartum cows increased blood glucose, decreased blood ketones and reduced the incidence of ketosis. Niacin flow to the small intestine and its absorption from the small intestine increased with niacin supplementation. Supplemental niacin prevented the postpartum decrease in red blood cell niacin observed in control cows.     

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.     

Sulfur-associated polioencephalomalacia in cattle grazing plants in the Family Brassicaceae

Polioencephalomalacia was diagnosed histologically in cattle from two herds on the Darling Downs, Queensland, during July–August 2007. In the first incident, 8 of 20 18-month-old Aberdeen Angus steers died while grazing pastures comprising 60% Sisymbrium irio (London rocket) and 40% Capsella bursapastoris (shepherd's purse). In the second incident, 2 of 150 mixed-breed adult cattle died, and another was successfully treated with thiamine, while grazing a pasture comprising almost 100% Raphanus raphanistrum (wild radish). Affected cattle were either found dead or comatose or were seen apparently blind and head-pressing in some cases. For both incidents, plant and water assays were used to calculate the total dietary sulfur content in dry matter as 0.62% and 1.01% respectively, both exceeding the recommended 0.5% for cattle eating more than 40% forage. Blood and tissue assays for lead were negative in both cases. No access to thiaminase, concentrated sodium ion or extrinsic hydrogen sulfide sources were identified in either incident. Below-median late summer and autumn rainfall followed by above-median unseasonal winter rainfall promoted weed growth at the expense of wholesome pasture species before these incidents.