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     

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.     

Serum lipid peroxide and alpha-tocopherol concentrations and superoxide dismutase activity in captive bottle-nosed dolphins.

OBJECTIVE: To evaluate serum lipid peroxide (LPO) and alpha-tocopherol concentrations and superoxide dismutase (SOD) activity in captive bottle-nosed dolphins and to evaluate effects of storage on production of LPO in various marine fish. ANIMALS: 16 bottle-nosed dolphins. PROCEDURE: 8 dolphins (group A) were fed chub mackerel and herring (high fat) and arabesque greenling and banded blue-sprat (low fat); the other 8 dolphins (group B) were fed chub mackerel and Pacific saury (high fat) and shishamo smelt and Japanese horse mackerel (low fat). Each group had been on these respective diets for 3 years. Serum LPO and alpha-tocopherol concentrations, serum SOD activity, and superoxide production by neutrophils were measured. All types of marine fish were frozen at -20 C for 6 months, and concentrations of LPO were measured at various time points. RESULTS: Serum LPO concentrations in group-A dolphins were significantly higher than those in group B. Serum alpha-tocopherol concentrations and SOD activity in group A were significantly lower than those in group B. A significant negative correlation was found between serum LPO and alpha-tocopherol concentrations in all 16 dolphins. The LPO concentrations in mackerel and herring fed to group-A dolphins were higher than those of other fish. Concentrations of LPO in herring stored for 3 and 6 months at -20 C were higher than those in herring before freezing and in herring stored for 1 month. CONCLUSIONS AND CLINICAL RELEVANCE: Serum LPO and alpha-tocopherol concentrations in captive bottle-nosed dolphins may be strongly influenced by high amounts of polyunsaturated fatty acid and LPO found in marine fatty fishes. High concentrations of serum LPO, as found in group-A dolphins, were associated with decreased antioxidative states. Monitoring of serum LPO and alpha-tocopherol concentrations and serum SOD activity may be useful for the management of captive marine mammals.     

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.     

Bacteriological quality of raw materials used in Finnish mink feed.

Mink feed raw materials were analyzed for total bacterial count, the number of faecal streptococci, the coliform count, the number of haemolytic bacteria and the number of sulphite reducing bacteria. The investigation comprised samples from the following raw materials: four slaughter-house offal products, preserved and unpreserved slaughter blood, Baltic herring, cod filletting offal, fish silage, blood meal, fish meal, meat-bone meal, protein concentrate, brewer’s yeast and cereal feed.The slaughter-house offals and unpreserved slaughter blood had the poorest quality, in terms of all the bacterial types for which the samples were analyzed. There were statistically significant differences in bacterial contents between slaughter-house offals from different sources. The preserved slaughter blood had significantly lower bacterial contents as compared to the unpreserved slaughter blood. Single samples of the cod filletting offal, Baltic herring and the blood meal had relatively high total bacterial counts, but the specified mean bacterial counts were relatively low. The bacterial counts for the rest of the investigated raw materials were relatively low.     

Clostridium botulinum in fish.

1407 fish caught in Scandinavian waters, the North Sea and the North Atlantic have been examined for the presence of Cl. botulinum. The incidence in gut samples expressed as percentage of fish tested was generally highest in fish from Scandinavian coastal waters and the Baltic Sea (4--43%), decreasing in fish from the North Sea (0--8%), and the organism was practically absent in fish from the North Atlantic. When gut samples were examined, the incidence was highest in demersal fish (cod and flatfish) as compared with pelagic fish (herring). The latter fish species were mainly contaminated on outer surfaces and gills. Only type E was detected in this survey, and Cl. botulinum was not detected in any wild fresh water fish. It is suggested that type E spores may originate in the sea bed and that they be spread by fish and water currents.     

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.     

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 (>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.     

Sequential pathology after experimental infection with marine viral hemorrhagic septicemia virus isolates of low and high virulence in turbot (Scophthalmus maximus L)

Three marine viral hemorrhagic septicemia virus isolates were used to bath challenge turbot with the purpose of studying mortality and the pathology and antigen distribution over time. Two high-virulence isolates, 860/94, 4p168 and a low-virulence isolate 1p3 from a Baltic Sea herring were used. Organ samples were collected sequentially at 2, 4, 7, 10, 15, 20, 25, and 45 days postinfection. Specimens were processed for virology, histopathology, and immunohistochemistry. Organs during the early stages of infection (from 2 to 7 days) had virus isolation from all three groups only on day 7. Virus titer in kidney and heart sampled at day 25 was higher for the two virulent isolates compared with the low-virulence isolate. The viral distribution in situ of the two more virulent isolates from turbot (860/94) and herring (4p168) resembled viral hemorrhagic septicemia in rainbow trout with regard to the target organs. Early infection of endothelial cells in both kidney and heart was observed. Accumulated mean mortality was 41.5% for the turbot isolate 860/94, 48% for the herring isolate 4p168, and 3.5% for the herring isolate 1p3. This study revealed that the isolates from turbot (860/94) and herring (4p168) induced significantly higher mortality compared with the virus-free control and the herring isolate (1p3). The onset of mortality is markedly later in turbot compared with what is seen in rainbow trout.     

Effect of various electric field strengths and current durations on stunning and spinal injuries of Atlantic herring

The aim of this study was to evaluate the effect of electric field strength and current duration on wild-caught Atlantic herring Clupea harengus stunned with sinusoidal 50-Hz AC in seawater. The fish were exposed to electric field strengths ranging from 16 to 142 V/m and current durations from 1 to 12 s. We recorded the elapsed times between the point at which each fish became unconscious and the points at which it resumed normal behavioral functions. We also investigated injuries such as broken spinal columns and hematomas after the fish were filleted. The threshold electric field strength required to stun all of the fish to unconsciousness was 33 V/m for 1 s. The duration of the unconscious condition increased as both electric field strength and current duration increased. Of a total of 260 Atlantic herring, 60% had broken spinal columns. The proportion of fish with fractured spines was independent of field strength and duration, but the number of fractures per fish increased with field strength. We conclude that electrical stunning would promote the welfare of Atlantic herring that are landed alive but negatively affect fillet quality owing to hematomas associated with the fractures.     

Evaluation of emamectin benzoate for the control of experimentally induced infestations of Argulus sp. in goldfish and koi carp

The effect of 0.2% emamectin benzoate (SLICE; Intervet/ Schering-Plough Animal Health, Roseland, New Jersey) administered in top-dressed, pelleted commercial fish feed was evaluated for control of freshwater Argulus sp. in goldfish Carassius auratus and koi carp, a variant of common carp Cyprinus carpio, in freshwater aquaria at 24-25 degrees C. Sixteen individually housed goldfish were each exposed to 37 Argulus. The number of fish lice attached to each fish at the start of the experiment was not determined; however, the total number of motile fish lice in each aquarium (on fish and in the water) was determined at the start and end of each experiment. Eight goldfish were fed the control diet (0 microg x kg fish biomass(-1) x d(-1)) and eight were fed the medicated diet (50 microg x kg fish biomass(-1) x d(-1)) for seven consecutive days. After treatment, fish louse infestation in controls was 20.5 +/- 1.5 (mean +/- SE) lice per fish. No Argulus were found on fish in the treated group. In a separate experiment, 10 individually housed koi were each exposed to 128 Argulus. Five koi were fed the control diet and five were fed a low-dose medicated diet (5 microg x kg fish biomass(-1) x d(-1)) for 7 d. After treatment, fish louse infestation among the controls was 14.6 +/- 3.8 lice per koi. No Argulus were found on koi in the treated group. Hence, a 7-d regimen of oral emamectin benzoate controlled experimental infestation of Argulus when administered to goldfish at 50 microg x kg fish biomass(-1) x d(-1) and to koi at 5 microg x kg fish biomass(-1) x d(-1).     

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.     

North American Strain of Viral Hemorrhagic Septicemia Virus is Highly Pathogenic for Laboratory-Reared Pacific Herring

Abstract

Specific-pathogen-free Pacific herring Clupea pallasi were reared in the laboratory from eggs and then challenged at 5, 9, and 13 months of age by waterborne exposure to low (10^1.5–2.5 plaque-forming units [PFU] per milliliter), medium (10^3.5–4.5 PFU/mL), or high (10^5.5–6.5 PFU/mL) levels of a North American isolate of viral hemorrhagic septicemia virus (VHSV). The fish were extremely susceptible to the virus, showing clinical disease, mortality approaching 100%, and only a limited increase in resistance with age. Mortality began 4–6 d after exposure and peaked at approximately day 7 in fish exposed to high levels of virus. Whereas the mean time to death showed a significant dose response (P < 0.001), the percent mortality and virus titers in dead fish were generally high in all groups regardless of initial challenge dose. External signs of disease were usually limited to 1–2-mm hemorrhagic areas on the lower jaw and isthmus and around the eye, but 2 of 130 infected fish exhibited extensive cutaneous hemorrhaging. Histopathologic examination of tissues from moribund fish sampled at 2–8 d after exposure revealed multifocal coagulative necrosis of hepatocytes, diffuse necrosis of interstitial hematopoietic tissues in the kidney, diffuse necrosis of the spleen, epidermis, and subcutis, and occasional necrosis of pancreatic acinar cells. Virus titers in tissues of experimentally infected herring were first detected 48 h after exposure and peaked 6-8 d after exposure at 10^7.7 PFU/g. Fish began shedding virus at 48 h after exposure with titers in the flow-through aquaria reaching 10^2.5 PFU/mL at 4–5 d after exposure, just before peak mortality. When the water flow was turned off for 3 h, titers in the water rose to 10^3.5 PFU/mL, and the amount of virus shed by infected fish (on average, greater than 10^6.5 PFU/h per fish) appeared sufficient to sustain a natural epizootic among schooling herring. Taken together, these data suggest that VHSV could be a significant limiting factor for populations of Pacific herring.     

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.     

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.     

Isolation of antibacteriologically active compounds from species of the Cottidae family

Antibacteriologically active compounds were isolated from the skin of several species of the fish family Cottidae. Suitable samples were obtained from species living in the Pacific, lakes in North America and the Fenno-Scandinavian peninsula as well as the Baltic sea. The compounds isolated from the skin of Triglops quadricornis (fourhorn sculpin) from the Baltic sea were particularly studied. The activity was partially characterised by chemical and biochemical investigations and the susceptibility of several human and fish pathogens described. The substance or substance(s) are of a high molecular mass, resist proteolytic and glycolytic enzymes, are heat and pH sensitive and water soluble.     

Reproductive efficiency and maternal-offspring transfer of gossypol in rainbow trout (Oncorhynchus mykiss) fed diets containing cottonseed meal.

In a preceding study, complete substitution of fish meal protein with cottonseed meal (CM) protein did not affect the survival or growth rate of adult rainbow trout over a 6-mo period. Gossypol, a naturally occurring compound in cottonseeds, has an antifertility effect in terrestrial animals, but information regarding salmonid fish is lacking. Female rainbow trout in this experiment were fed diets with either 0, 25, 50, 75, or 100% (diets 1 to 5) of the fish meal protein replaced with CM protein until first maturation and spawning to study long-term effects on growth and reproduction. Feeding diets containing CM over a total period of 10 mo did not result in differences in growth and mortality compared to the control group (P > 0.05). Increased CM incorporation levels resulted in decreased (P < 0.05) blood hemoglobin (10.6 +/- 1.3, 8.4 +/- 1.8, 7.3 +/- 1.1, 6.9 +/- 0.8, and 5.6 +/- 1.4 g/dL) and hematocrit (49.6 +/- 3.9, 38.5 +/- 9.3, 34.4 +/- 3.7, 34.8 +/- 4.9 and 28.0 +/- 6.8%) levels in diets 1 to 5, respectively. The CM incorporation level had no effect (P > 0.05) on the number of eggs produced per female but led to a reduction (P < 0.05) in egg weight. Eyed stage survival of embryos was low in all dietary groups and did not show differences (P > 0.05). However, an increasing CM incorporation level led to a linear increase (P < 0.05) in the number of females that produced no viable embryos (23.1, 37.5, 42.9, 60.0, and 71.4%). Gossypol in the diet was absorbed by the female trout and transferred to the eggs (0, 2.2 +/- 0.5, 6.7 +/- 1.6, 10.6 +/- 4.2, and 20.0 +/- 2.6 micrograms/g in diets 1 to 5, respectively). A high concentration of gossypol remained in the juveniles at the swim-up stage (endogenous yolk-absorbed) (0.6 +/- 0.3, 2.4 +/- 0.3, 3.4 +/- 0.0, and 4.7 +/- 1.0 micrograms/g, diets 2 to 5, respectively). The findings suggest that replacement of the dietary fish meal protein with CM protein has no effect on fish growth and mortality but may lead to a reduction in reproductive performance in female rainbow trout.     

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.     

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.