Ryegrass Staggers: The Presence of Lolitrem Neurotoxins in Perennial Ryegrass Seed

Abstract

Extract

Sir:- Recently, two potent neurotoxins named lolitrem A and lolitrem B were isolated from herbage collected from pastures on which the livestock disorder ryegrass staggers (RGS) occurred. et al.⁽¹⁾ Gallagher, R.T., White, E.P. and Mortimer, P.H. 1981. Ryegrass staggers: Isolation of potent neurotoxins lolitrem A and lolitrem B from staggers-producing pastures. N.Z. vet. J., 29: 189–190. [Taylor &; Francis Online], [Web of Science ®] , [Google Scholar] We now wish to report that samples of perennial rygrass (Loliun yeretzrze L.) seed obtained from several locations in New Zealand contained potent neurotoxins which include lolitrem A and lolitrem B. Further, when pellets made from lolitrem neurotoxin-containing seed were fed to sheep, symptoms indistinguishable from those of severe, natural RGS were induced in four sheep, and mild symptoms in three sheep.     

Lolitrem B residue in fat tissues of cattle consuming endophyte-infected perennial ryegrass straw.

Lolitrems are neurotoxins found in endophyte-infected perennial ryegrass. Lolitrems, primarily lolitrem B, are the causative agents of ryegrass staggers in livestock. To guarantee the safety of meat produced from cattle consuming endophyte-infected perennial ryegrass, lolitrem B concentrations in tissues of Japanese Black cattle were determined using high-performance liquid chromatography. Lolitrem B was not detected in muscle, liver, kidney, or cerebrum of a Japanese Black cow with signs of ryegrass staggers. In contrast, perirenal fat contained 210 ppb lolitrem B. Three cows that received half as much perennial ryegrass straw as the cow with ryegrass staggers showed no clinical signs of ryegrass staggers. However, low concentrations of lolitrem B (less than 150 ppb) were detected in their fat tissue. These observations indicate that human exposure to the neurotoxic effect of lolitrem B through beef is unlikely. The amount of lolitrem B consumed by cattle can be estimated by the determination of lolitrem B in fat tissue.     

The evaluation of endophyte toxin residues in sheep fat

Abstract

AIM: To monitor changes in concentrations of lolitrem B and epoxy-janthitrems in the fat of sheep grazing perennial ryegrass infected with wild-type- and AR37-endophyte, respectively, during the time of year when ryegrass staggers would be expected to be observed.

METHODS: Ten 5-month-old lambs with no previous exposure to endophytes were grazed on either wild-type (containing lolitrem B, n = 5) or AR37 (containing epoxy-janthitrems, n = 5) endophyte-infected perennial ryegrass (Lolium perenne L.) pastures between October 2008 and June 2009. Animals were regularly assessed for ryegrass staggers using the Keogh scale (0 = no signs, 5 = severe tremors). When a score of > 3.5 was observed animals were removed from the treatment pastures for 1 month. Fat biopsy samples were taken from each animal at approximately monthly intervals and analysed for endophyte metabolites using high performance liquid chromatography (HPLC) methods developed during this study. Regular herbage samples were also taken and concentrations of endophyte metabolites measured.

RESULTS: Efficient and reproducible methods to analyse both lolitrem B and epoxy-janthitrems in fat were developed. Concentrations of lolitrem B and epoxy-janthitrems in herbage and in sheep fat increased from late November to peak in mid-February. Ryegrass staggers was observed in both groups of sheep at this time. Following 1 month of grazing non-infected pasture mean concentrations in fat of lolitrem B decreased by 43% from 61.8 to 35.3 ppb, and of epoxy-janthitrems by 38% from 1032.0 to 639.5 ppb. Maximum concentrations in herbage of epoxy-janthitrems (35.7 ppm) were higher than of lolitrem B (3.4 ppm), but signs of staggers were less severe in sheep grazing pasture containing the former compared with the latter (median Keogh scores in late February were 2 and 3, respectively), consistent with epoxy-janthitrems being low potency toxins.

CONCLUSION: This study demonstrated that concentrations of epoxy-janthitrems and lolitrem B in sheep fat increased quickly during the initial phase of the study when concentrations in pasture increased, and decreased when animals were removed from pastures containing these compounds. These data will be used in the risk assessment of the endophyte metabolites.     

Flow-mediated K+ secretion in horses intoxicated with lolitrem B (perennial ryegrass staggers)

AIM: To investigate the effects of lolitrem B intoxication on renal K⁺ secretion in response to increased tubular flow rates.

METHODS: Results are derived from a repeated measure pilot study of seven horses fed non-perennial ryegrass feed for a week prior to exposing them to perennial ryegrass seed and hay that contained an average of 2 ppm lolitrem B. At the end of the control and treatment period frusemide (1 mg/kg I/V) was administered and serial fractional excretion of K⁺(FEK⁺) and fractional excretion of Na⁺(FENa⁺) calculated. Baseline concentration of aldosterone in plasma, serum K⁺concentration and feed K⁺ concentration were also compared.

RESULTS: Key findings included a reduced change in FEK⁺ from 0 to 15 minutes in response to frusemide administration (p=0.022, Wilcoxon signed-rank test) and a reduced baseline concentration of aldosterone in plasma (p=0.022, Wilcoxon signed-rank test) during the treatment period compared with the control.

CONCLUSIONS: Results suggest that lolitrem B intoxication reduced flow-mediated K⁺ secretion and interfered with aldosterone production or secretion. However, further investigation is required to validate these findings and to further elucidate the underlying pathophysiology.

CLINICAL RELEVANCE: Lolitrem B intoxication in horses may cause disruption to electrolyte handling in addition to neurological deficits.     

Ergovaline does not alter the severity of ryegrass staggers induced by lolitrem B

AIM: To investigate a possible interaction between lolitrem B and ergovaline by comparing the incidence and severity of ryegrass staggers in sheep grazing ryegrass (Lolium perenne) containing lolitrem B or ryegrass containing both lolitrem B and ergovaline.

METHODS: Ninety lambs, aged approximately 6 months, were grazed on plots of perennial ryegrass infected with either AR98 endophyte (containing lolitrem B), standard endophyte (containing lolitrem B and ergovaline) or no endophyte, for up to 42 days from 2 February 2010. Ten lambs were grazed on three replicate plots per cultivar. Herbage samples were collected for alkaloid analysis and lambs were scored for ryegrass staggers (scores from 0–5) weekly during the study. Any animal which was scored ≥4 was removed from the study.

RESULTS: Concentrations of lolitrem B did not differ between AR98 and standard endophyte-infected pastures during the study period (p=0.26), and ergovaline was present only in standard endophyte pastures. Ryegrass staggers was observed in sheep grazing both the AR98 and standard endophyte plots, with median scores increasing in the third week of the study. Prior to the end of the 42-day grazing period, 22 and 17 animals were removed from the standard endophyte and AR98 plots, respectively, because their staggers scores were ≥4. The cumulative probability of lambs having scores ≥4 did not differ between animals grazing the two pasture types (p=0.41).

CONCLUSIONS AND CLINICAL RELEVANCE: There was no evidence for ergovaline increasing the severity of ryegrass staggers induced by lolitrem B. In situations where the severity of ryegrass staggers appears to be greater than that predicted on the basis of concentrations of lolitrem B, the presence of other tremorgenic alkaloids should be investigated.     

Evaluation of perennial ryegrass straw as a forage source for ruminants

Two experiments were conducted to evaluate perennial ryegrass straw as a forage source for ruminants. Experiment 1 evaluated digestion and physiological variables in steers offered perennial ryegrass straw containing increasing levels of ergot alkaloid, lolitrem B. Sixteen ruminally cannulated Angus x Hereford steers (231+/-2 kg BW) were blocked by weight and assigned randomly to one of four treatments. Steers were provided perennial ryegrass straw at 120% of the previous 5-d average intake. Before straw feeding, soybean meal was provided (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Low (L) and high (H) lolitrem B straws (<100 and 1,550 ppb, respectively; DM basis) were used to formulate treatment diets: 100% L; 67% L:33% H; 33% L:67% H; 100% H (DM basis). Intake and digestibility of DM and OM, and ruminal pH, total VFA, and NH3-N were not affected by increasing lolitrem B concentration. Ruminal indigestible ADF (IADF) fill increased linearly (P = 0.01) and IADF passage rate decreased linearly (P = 0.04) as lolitrem B increased. Experiment 2 evaluated performance and production by 72 Angus x Hereford cows (539+/-5 kg BW) consuming perennial ryegrass straw containing increasing lolitrem B during the last third of gestation. Cows were blocked by body condition score and randomly assigned to one of three treatments. Cows were provided perennial ryegrass straw ad libitum and supplemented with soybean meal (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Mixtures of a L and H lolitrem B straw (467 and 2,017 ppb, respectively) were used to formulate treatment diets: 100% L, 50% L:50% H, 100% H (DM basis). Thirteen of 24 cows on the 100% H treatment exhibited signs of ryegrass staggers and were removed from the study; nevertheless, lolitrem B concentration did not influence pre- or postcalving weight or body condition score change. These data suggest that feeding perennial ryegrass straw containing up to 1,550 ppb lolitrem B (DM basis) did not adversely affect nutrient digestion or physiological response variables in steers. However, providing straw with a lolitrem B concentration of approximately 2,000 ppb (DM basis) resulted in 54% of cows exhibiting signs of ryegrass staggers. These data suggest that blending straws with a high (>2,000 ppb) and low (<500 ppb) concentration of lolitrem B can be a successful management practice.     

Tremorgenic mycotoxins paxilline, penitrem and lolitrem B, the non-tremorgenic 31-epilolitrem B and electromyographic activity of the reticulum and rumen of sheep

The mycotoxic tremorgens penitrem, paxilline and lolitrem B had profound effects on electromyographic (EMG) activity of smooth muscle of the reticulorumen in conscious sheep, with a similar time course of action to their respective characteristic effects on the induction (1 to 2, 15 to 20 and 20 to 30 minutes) and the duration (1 to 2, 1 to 2 and 8 to 12 hours) of tremoring. Responses to penitrem revealed a greater sensitivity of smooth muscle than skeletal muscle. Effects included an inhibition of the vagally-dependent cyclical A and B sequences of contraction of the reticulorumen, an increase in their amplitude and an excitation of local intrinsic activity contributing to elevated baselines and the occurrence of chaotic activity of the reticulum. The excitatory local effects were partially blocked by atropine, indicating that stimulation of muscarinic cholinoceptors was involved. Increased local activity may mediate a reflex inhibition of cyclical contractions. A non-tremorgenic isomer of lolitrem B (31-epilolitrem B) had no effect on the reticulorumen. The intensity and duration of the effects of lolitrem B, up to 12 hours, indicate that severe disruption of digestion may occur in animals grazing endophyte-infected pasture.     

Correlation of ergovaline and lolitrem B levels in endophyte-infected perennial ryegrass (Lolium perenne).

The varieties of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotiphodium lolii contain several classes of toxic alkaloids, including ergopeptide alkaloids and lolitrem alkaloids. Lolitrem B, a potent tremorgen, is generally considered to be the predominant alkaloid in endophyte-infected perennial ryegrass. Ergovaline, a vasoconstrictor normally associated with endophyte-infected tall fescue (Festuca arudinacea), is also present in endophyte infected perennial ryegrass. Clinical signs of animals ingesting endophyte-infected perennial ryegrass are consistent with the presence of lolitrem B. However, clinical signs normally associated with ergovaline poisoning are not usually observed in animals ingesting endophyte-infected perennial ryegrass. A survey was conducted to quantitate both lolitrem B and ergovaline in 459 perennial ryegrass straw samples received at the Oregon State University College of Veterinary Medicine. Samples were analyzed for each alkaloid using separate high-performance liquid chromatography analyses. A strong positive correlation between the 2 alkaloids (r2 = 0.7335) was observed, especially in the samples containing <3,000 ppb (ng/g) lolitrem B. The threshold levels above which clinical signs typically occur are 2,000 ppb lolitrem B and 300-400 ppb ergovaline. All of the samples analyzed contained <425 ppb ergovaline.     

The effect of glyphosate, paraquat and paclobutrazol on lolitrem B levels in endophyte-infected perennial ryegrass

Two herbicides (glyphosate and paraquat) and a plant growth regulator (paclobutrazol) were applied to endophyteinfected (Acremonium lolii) perennial ryegrass swards. Subsamples of these swards were then chemically analysed at intervals up to 28 days later for lolitrem B, the compound responsible for perennial ryegrass staggers in domestic livestock. Glyphosate and paclobutrazol had no effect on lolitrem B concentrations. Paraquat applications decreased lolitrem B concentrations in the herbage. Because none of the chemicals tested increased the concentration of lolitrem B in the herbage, they are unlikely to be directly implicated in perennial ryegrass staggers in grazing animals.     

Preliminary investigation on rodent–ectoparasite associations in the highlands of Tigray,Northern Ethiopia: implications for potential zoonoses

We studied associations between rodents and their arthropod ectoparasites in crop fields and household compounds in the highlands of Tigray, Northern Ethiopia. Ectoparasite infestation indices, such as percent infestation, mean abundance, prevalence and host preferences, were calculated for each taxon. In total, 172 rodents from crop fields and 97 from household compounds were trapped. Rodent species and numbers trapped from the crop fields and household compounds were Mastomys awashensis (Lavrenchenko, Likhnova & Baskevich, 1998) (88 and 44), Arvicanthis dembeensis (Ruppel, 1842) (63 and 37) and Acomys sp. (21 and 16), respectively. A total of 558 insects and acarids (belonging to 11 taxa) were recovered from the rodents trapped in the crop fields, and 296 insects and acarid (belonging to 6 taxa) from the rodents trapped in the household compounds. Approximately 66% of the rodents trapped from the crop fields and 47% of those trapped from the household compounds were infested with ectoparasites. Laelaps sp. (64.9%) and Xenopsylla sp. (20.6%) comprised the highest proportion of the ectoparasites recovered in the crop fields, and the same ectoparasites, but in reverse order, comprised the highest proportions in the household compounds (Xenopsylla [50.3%] and Laelaps sp. [29%]). Our study revealed that crop fields and household compounds in the highlands share similar rodents and several ectoparasites. Furthermore, at least 1 of the rodent species and some of the ectoparasites identified in this study were reported to have posed medical and veterinary threats in other parts of Ethiopia and neighboring countries.     

Stable isotope analysis of diet confirms niche separation of two sympatric species of Namib Desert lizard

We used stable isotopes of carbon and nitrogen to study the trophic niche of two species of insectivorous lizards, the Husab sand lizard Pedioplanis husabensis and Bradfield's Namib day gecko living sympatrically in the Namib Desert. We measured the δ¹³C and δ¹⁵N ratios in lizard blood tissues with different turnover times (whole blood, red blood cells and plasma) to investigate lizard diet in different seasons. We also measured the δ¹³C and δ¹⁵N ratios in available arthropod prey and plant tissues on the site, to identify the avenues of nutrient movement between lizards and their prey. Through the use of stable isotope mixing models, we found that the two lizard species relied on a largely non‐overlapping but seasonally variable array of arthropods: P. husabensis primarily fed on termites, beetles and wasps, while R. bradfieldi fed mainly on ants, wasps and hemipterans. Nutrients originating from C₃ plants were proportionally higher for R. bradfieldi than for P. husabensis during autumn and late autumn/early winter, although not summer. Contrary to the few available data estimating the trophic transfer of nutrients in ectotherms in mixed C₃ and C₄/crassulacean acid metabolism (CAM) plant landscapes, we found that our lizard species primarily acquired nutrients that originated from C₄/CAM plants. This work adds an important dimension to the general lack of studies using stable isotope analyses to estimate lizard niche partitioning and resource use.     

Orbit orientation and eye morphometrics in giraffes (Giraffa camelopardalis)

Giraffe are thought to have excellent vision. We measured eye size, orbit orientation and retina surface area in 27 giraffes of both sexes ranging in age from neonates to mature adults (>10 yrs), to assess how it changes with growth, whether their eye anatomy correlates with their apparently excellent vision and lifestyle, and we have compared our findings with those for other large mammals to assess whether giraffe eye anatomy is unique. We found that giraffe eye volume increases from 33 cm³ at birth to approximately 65 cm³ in adults. The focal (axial) length increases from c. 40 to 48 mm in adults and retina surface area from c. 3000 mm² at birth to 4320 mm² in adults. The orbital axis angle at birth is c. 73° and the horizontal visual field mainly monocular and panoramic. With age the axis angle becomes more acute to c. 50° in adults and the visual field more binocular, changes that occur concurrently with increasing neck length. These results show that the giraffe eye and retinal surface area are larger than in all other ungulates, and their visual fields more binocular, attributes which are consistent with the idea that they have excellent vision.     

Comparative study of the effects of acepromazine and structurally related compounds on the TNF-α release by monocytes stimulated by Chlamydia pneumoniae

Acepromazine (ACP), a member of the phenothiazine family, has antioxidant properties and interacts with reactive oxygen species produced by stimulated neutrophils ( Serteyn et al. 1999 ). We found that ACP reduced the differentiation of monocytes induced by an overnight incubation with a crude Chlamydia pneumoniae extract ( Serteyn et al. 2001 ). The same model was used to test the effects of phenothiazines on the TNF‐α release by activated monocytes. A crude Chlamydia pneumoniae extract was obtained by mechanical disruption and centrifugation (1 minute, 1500 r.p.m.) of 78 hours infected McCoy cells. Monocytes (THP1 cell line; 2 × 10⁶ cells by assay) were incubated overnight with 30 µL of Chlamydia pneumoniae crude extract (equivalent to an endotoxin charge of 3.5 pg) in the presence or absence of phenothiazines (from 10^?6 to 10^?4 M) ( Mouithys‐Mickalad et al. 2001 ). For estimation of TNF‐α release, the supernatants were collected, centrifuged (to eliminate the undifferentiated monocytes) and used for TNF‐α measurements (n = 6) (Quantikine HS human TNF‐α, R&D Systems, UK). Acepromazine was compared to other phenothiazines (chlorpromazine, trifluoperazine) or to structural analogues of phenothiazines (phenoxazine, thioxanthen‐9‐one and methylene blue). For each assay, cytotoxicity was evaluated by microscopic examination and blue trypan exclusion method. Mean values of TNF‐α were compared by a Student t‐test (p < 0.05). TNF‐α release by Chlamydia‐treated THP1 was significantly decreased by ACP in a dose‐dependent manner, 378 ± 30, 209 ± 38 and 189 ± 35 ng mL^?1 for 10^?6, 10^?5 and 10^?4 M compared to the control values 385 ± 9 ng mL^?1. Similar inhibitions of TNF‐α release were obtained with trifluoperazine (313 ± 25 and 265 ± 14 ng mL^?1 at 10^?6 and 10^?5 M) and chlorpromazine (323 ± 29 and 227 ± 13 ng mL^?1 at 10^?6 and 10^?5 M), but at 10^?4 M, these two drugs were cytotoxic. The other structurally parent compounds increased significantly the TNF‐α production: 630 ± 46 and 468 ± 60 ng mL^?1 for thioxanthen‐9‐one and 547 ± 17 and 331 ± 111 ng mL^?1 for methylene blue at 10^?5 and 10^?6 (M). At 10^?4 M, the two compounds were cytotoxic. Phenoxazine increased the TNF‐α production, slightly at 10^?6 and 10^?5 M (444 ± 39 and 424 ± 16 ng mL^?1, respectively) and significantly at 10^?4 M (959 ± 30 ng mL^?1). Further studies are needed to verify if the inhibition of TNF‐α release by some phenothiazines could be linked to a reduction of the signal transduction, especially the NF‐κB pathway. These results could be interesting for the anaesthesia or treatment of animals suffering from a systemic inflammatory reaction.     

Growth Inhibition of Selected Aquatic Bacteria by Tannic Acid and Related Compounds

Abstract

Tannic acid, propyl gallate, methyl gallate, and gallic acid were tested for their inhibitory effects on selected aquatic microorganisms by the well assay technique. Tannic acid, propyl gallate, and methyl gallate in deionized water inhibited the growth of Aeromonas hydrophila, A. sobria, Edwardsiella iclaluri, E. tarda, Pseudomonas fluorescens, and Escherichia coli, but gallic acid did not. When 500-μg/mL concentrations of these four compounds were tested in sterilized fish pond water at pH 7.0 and with a low bacterial inoculum of 10³–10⁴ colony-forming units (CFU) per milliliter, they inhibited the growth of P. fluorescens and (except for tannic acid) E. coli. Pseudomonas fluorescens inoculated at 10³–10⁴ CFU/mL in pond water was inhibited by methyl gallate, propyl gallate, and tannic acid concentrations as low as 50 μg/mL, but a gallic acid contration of 100 μg/mL was required for inhibition. Escherichia coli was inhibited by methyl gallate and propyl gallate at 250 μg/mL and by gallic acid at 500 μg/mL, but it was not inhibited by tannic acid at concentrations up to 500 μg/mL in water of pH 7.0. Tannic acid (500 μg/mL) did inhibit E. coli growth at pH 4.5.     

Equine applications of magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) is a rapidly developing technique on the threshold of several equine applications. This technique measures phosphorus and hydrogen compounds non-invasively, in real time and in vivo. It has great potential as a powerful new laboratory and clinical tool. There are 2 separate modalities: imaging of tissues (MRI) from the resonance of protons [¹H] in soft tissues, and as a chemical spectrometer (MRS).A magnetic resonance spectrometer can monitor the dynamics of muscle energetics during exercise; measuring ATP, PCr, Pi and H⁺ directly. ADP can also be derived from the Pi/PCr ratio. In principle, muscle lactate may also be obtained from ¹H and muscle glycogen from ¹³C. Analysis of Pi/PCr as a function of work rate provides a direct measure of biochemical efficiency and capacity.Typically an animal is placed in the bore of a cylindrical superconducting magnet. A radio-frequency coil is placed over the muscle to detect molecular resonance within the tissue. Signal spectra are resolved by Fourier transformation, then computer enhanced so that compounds of known resonance can be quantified. This is more relevant to cellular function than conventional biochemistry, because with MRS only metabolically active (unbound) molecules are included. Furthermore MRS avoids biopsy and the consequent disruption of cells. Horses are ideal for spectroscopy since inherent problems of slow data acquisition and poor signal-to-noise markedly improves with their higher muscle mass and mitochondrial density. This device has great potential applications to equine practice; although it is still under development, and a large-scale system has yet to be constructed. It is also, with horses, a major technical and financial challenge.     

Voluntary intake,chemical composition and <Emphasis Type="Italic">in vitro</Emphasis> digestibility of fresh forages fed to Guinea pigs in periurban rearing systems of Kinshasa (Democratic Republic of Congo)

Bindelle, J., Ilunga, Y., Delacollette, M., Muland Kayij, M., Umba di M’Balu, J., Kindele, E. and Buldgen, A. Voluntary intake, chemical composition and in vitro digestibility of fresh forages fed to Guinea pigs in periurban rearing systems of Kinshasa (Democratic Republic of Congo). Tropical Animal Health and Production. The daily voluntary intake (DVI) of Guinea pigs (GP) fed 15 fresh forages used in periurban rearing systems of Kinshasa (Democratic Republic of Congo) was investigated. In order to determine the best forages combination for GP diet, DVI was compared to their nutritional value measured in vitro using 1) a pepsin-pancreatin hydrolysis, 2) an gas fermentation test on the hydrolysed residues with an inoculum prepared from GP faeces, and 3) the chemical composition of the offered feeds and the hydrolysis residues. The forages ranking based on the DVI was correlated to the NDF content, but not to their nutritional values determined in vitro. According to their high DVI (from 4.23 to 7.75 g/kg liveweigth), and their valuable in vitro nutritional values (crude protein ranging from 261 to 279 g crude protein kg⁻¹DM, pepsin-pancreatin digestibilities of the dry matter from 0.55 to 0.59 and final gas production from 170 to 196 l kg⁻¹DM), Desmodium intortum, Euphorbia heterophylla or Amaranthus hybridus, can be suggested to the farmers to complement the usual diet distributed to the GP based on Panicum maximum.     

Reproduction traits of heterozygous myostatin knockout sows crossbred with homozygous myostatin knockout boars

Few studies exist on homozygous myostatin gene mutant (MSTN^−/−) pigs, especially on their reproductive ability. We have previously shown that semen quality of homozygous MSTN^−/− boars is comparable to that of wild type (WT). However, no data exist on the reproductive ability of heterozygous MSTN gene mutant (MSTN^+/−) sows. The present study highlights showed that the heterozygous MSTN^+/− sows have delayed pubertal age than WT sows (255.80 ± 6.79 versus 191.10 ± 3.42, respectively). The number of services per pregnancy of heterozygous MSTN^+/− sows is significantly higher than that of WT sows (3.33 ± 0.43 versus 1.60 ± 0.25, respectively). Moreover, although heterozygous MSTN^+/− sows have natural reproduction ability, their litter size was significantly lower than that of WT sows (7.75 ± 0.44 versus 14.25 ± 0.60, respectively). Offsprings generated from heterozygous MSTN^+/− sow and homozygous MSTN^−/− boar were genotyped with the PCR and sequencing method to detect myostatin mutation and to identify whether the piglets are homozygous MSTN^−/− or heterozygous MSTN^+/−. The proportion of homozygous MSTN^−/− piglets was significantly lower than that of heterozygous MSTN^+/− piglets (2.50 ± 0.35 versus 5.25 ± 0.60, respectively). Furthermore, none of the sows presented dystocia, and the phenotype of heterozygous MSTN^+/− piglets was normal. However, 10% homozygous MSTN^−/− piglets died of dyspnoea within 2 hr after birth, 60% of homozygous MSTN^−/− piglets showed large tongues, and 50% had umbilical hernias. In summary, this study for the first time reports the reproduction traits of heterozygous MSTN^+/− sows crossbred with homozygous MSTN^−/− boars. This study will pave the way in a new direction for the breeding and development of super lean meat varieties in the future.     

Thysanoptera infestation on skin and periorbital cellulitis in ostriches (Struthio camelus) aged 14 months

AIM: To report the infestation of Thysanoptera (Limothrips denticornis) on ostriches (Struthio camelus) and to determine their relative density.

METHODS: A farm in Poland was studied on which ostriches aged 14 months were severely infested with L. denticornis (thrips). Thrips were collected and their density on the neck, torso and legs (10 cm²) of 85 ostriches determined at 0600, 1200 and 1800 h, respectively, over 7 days. At the same times, apparent densities of thrips/m² were determined in adjacent grassy areas (120 m²) on a muslin cloth impregnated with permethrin. Thrips were mounted onto slides in Hoyer's medium, for identification.

RESULTS: The apparent density of thrips was greatest at 1200 h, and was greater on the neck than the torso and legs (p=0.03). In adjacent surroundings, densities were also highest at 1200 h (mean 199 (SE 9.3) thrips/m²) and were similar to the maximum densities recorded on the necks of ostriches at that time (mean 205 (SE 6.4) thrips/m²). Ostriches engaged in excessive preening and attempted to scratch their head/neck with their feet. Numerous small, pale red papules were observed on the skin. Observations of periorbital cellulitis, conjunctivitis, blepharitis, watery discharge, inflammation, and sclerotic discolouration were noted in 65 ostriches.

CONCLUSION: Observations were commensurate with L. denticornis infestation.

CLINICAL RELEVANCE: Infestation may exacerbate stress levels and subsequently lead to a reduction in feed intake and performance. The infestation and subsequent irritation from thrips impacts negatively on the general health of ostriches by damaging skin and irritating eyes.     

The In Vitro Secretion of Acetylcholinesterase by Adult Stages of Heligmosomoides polygyrus: the Effects of Broad‐Spectrum Anthelmintics

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 ± 0.07 µmol min^?1 l^?1 mg^?1) than for female (0.56 ± 0.04 µmol min^?1 mg^?1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug‐free medium (RPMI medium supplemented with 0.5 % BSA) for 24 h or continuously exposed to the drugs in supplement‐free medium (24 h), the concentration‐ and time‐dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50 % (IC₅₀) relative to drug‐free controls were estimated. The IC₅₀ values ranged from 0.012 µM (IVM) to 2.96 µM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad‐spectrum anti‐nematodal activity is discussed.