Pharmacological evaluation of contractile activity of the dog heartworm <Emphasis Type="Italic">Dirofilaria immitis</Emphasis>

Effects of a variety of compounds on spontaneous contractile activity of whole, intact, adult canine heartworms (HW), which had been maintained in culture, were evaluated to improve understanding of the pharmacological sensitivities of this parasitic nematode. Acetylcholine, pilocarpine, imidazole, levamisole, and DL-tetramisole caused spastic paralysis. Gamma-aminobutyrate (GABA), the GABA-mimetic muscimol, the GABA amino transferase inhibitor 3-mercaptopropionic acid, fenthion, ketamine, levodopa, and salinomycin caused flaccid paralysis. Atropine and monensin had inhibitory effects. Neostigmine, the neuromuscular blocking agents decamethonium, succinylcholine, and D-tubocurarine, and the aminergic agents epinephrine, norepinephrine, and serotonin had little or no effect on contractile activity. Thiacetarsamide had a nonreversible, slow onset, inhibitory effect on contractile activity. Occurrence of spastic or flaccid paralysis was not correlated with gender or culture age and was never associated with the same compound. Submaximal stimulatory or inhibitory responses paralleled the type of maximal responses (spastic or flaccid paralysis) for most compounds. Concentration variations producing maximal effects suggested considerable variation in individual preparation sensitivity, which did not appear to involve cuticle defects or time in culture. Difference in gender sensitivity was noted only for levamisole, which caused greater stimulation of contractile activity in males than in females.     

Comparative in vitro effects of closantel and selected β-ketoamide anthelmintics on a gastrointestinal nematode and vertebrate liver cells

PNU-87407 and PNU-88509, β-ketoamide anthelmintics that are structurally related to each other and to the salicylanilide anthelmintic closantel, exhibit different anthelmintic spectra and apparent toxicity in mammals. The basis for this differential pharmacology was examined in experiments that measured motility and adenosine triphosphate (ATP) levels in larval and adult stages of the gastrointestinal nematode, Haemonchus contortus , and in a vertebrate liver cell line and mitochondria. PNU-87407 and PNU-88509 both exhibited functional cross-resistance with closantel in larval migration assays using closantel-resistant and -sensitive isolates of H. contortus . Each compound reduced motility and ATP levels in cultured adult H. contortus in a concentration- and time-dependent manner; however, motility was reduced more rapidly by PNU-88509, and ATP levels were reduced by lower concentrations of closantel than the β-ketoamides. Tension recordings from segments of adult H. contortus showed that PNU-88509 induces spastic paralysis, while PNU-87407 and closantel induce flaccid paralysis of the somatic musculature. Marked differences in the actions of these compounds were also observed in the mammalian preparations. In Chang liver cells, ATP levels were reduced after 3 h exposures to 0.25 μ M PNU-87407, 1 μ M closantel or 10 μ M PNU-88509. Reductions in ATP caused by PNU-88509 were completely reversible, while the effects of closantel and PNU-87407 were irreversible. PNU-87407, closantel and PNU-88509 uncoupled oxidative phosphorylation in isolated rat liver mitochondria, inhibiting the respiratory control index (with glutamate or succinate as substrate) by 50% at concentrations of 0.14, 0.9 and 7.6 μ M , respectively.     

Effect of Calea serrata Less. n-hexane extract on acetylcholinesterase of larvae ticks and brain Wistar rats

The stem bark of Acacia oxyphylla Graham ex Bentham is used as an anthelmintic by the natives of Mizoram (North-East India). Therefore, the aim of the study was to assess the effect of the active compound isolated from A. oxyphylla on the tegument of adult Raillietina echinobothrida and Ascaridia galli. The test parasites R. echinobothrida and A. galli were incubated in physiological buffered saline containing 0.0005, 0.001, 0.05, 0.1 and 1mg/ml of the isolated compound. The alterations in the tegument of the parasites post paralysis were examined using electron microscopes. The compound reduced the cestode's motility soon after incubation, but did not induce paralysis in the nematodes till about 11-14 h at highest concentration. The compound caused extensive digestion of cestode tegument as evident by electron microscopy. Disorganization of muscle bundles, loss of cell-cell contact, extreme vacuolization and oedema were some of the changes observed. Loss of cellular organelles combined with distortion of those present was markedly noted throughout the parasite tissue. Deformation and disorganization of epicuticle, disruption of mitochondrial and nuclear membrane were also observed in nematode exposed to the active compound of the plant. Substantial structural deformities in the treated parasites are indicative of an efficient vermicidal activity of the isolated compound against cestodes and nematodes.     

Contractile activity of cultured adult Dirofilaria immitis

A method for long-term maintenance of adult heartworms (HW) in culture for use in contractile activity studies was developed. Culture conditions included Eagle's minimum essential medium (MEM) containing Earle's balanced salt solution and MEM vitamins and supplemented with 10% horse serum, pH 7.6, 37 degrees C, and humidified 5% CO2:95% room air atmosphere. Motility was observed for up to 91 days. Reducing the culture atmosphere from 20% oxygen to 5% oxygen reduced acid production and survival to 28 days or less. Spontaneous contractile activity of adult HW coils (1 cm diameter) was measured using an isometric force displacement transducer system. Activity had an arrhythmic pattern of good magnitude that could be recorded after up to 50 days in culture for male HW and after up to 40 days in culture for female HW. Analyses of contractile activity included determination of its amplitude, frequency, contraction index, and basal tension. Amplitude for males (3.4 +/- 1.2 g) (mean +/- S.D.) was significantly greater (P < 0.02) than that for females (3.0 +/- 1.1 g), whereas frequency for females (8.2 +/- 2.3 min) was significantly greater (P < 0.03) than that for males (7.5 +/- 2.3 min). The contraction index for females was 16.7 +/- 13.7 mm/min and for males, 14.4 +/- 9.0 mm/min. The difference was not significant. The contraction index was based on line integration of the record of contractile activity. Amplitude and frequency of contractile activity for anterior segments (2.5 cm), suspended lengthwise, from cultured adult female HW, were not significantly different from results for coils, but the contraction index (34.5 +/- 33.8) was significantly higher (P < 0.01) indicating that the pattern of activity was more uniform in the segments. An applied basal tension of about 4 g was suitable for the coils, while a suitable basal tension for segments was about 1.5 g. For coils, amplitude, frequency, and contraction index increased significantly (P < 0.02) with increase in basal tension. Coils were not sensitive to changes in ionic, glucose, oxygen, and osmol concentrations, and pH (6.8-7.6) of the bathing solution. A pH of 8.0 markedly increased basal tension and contractile activity. At 20 degrees C contractile activity stopped. These effects of pH and temperature were reversible. The HW appeared resistant to changes in their environment. This is probably related to an efficient cuticular barrier. The culture and recording methods used in this study open improved opportunities for quantitative evaluation of responsiveness of HW to a variety of physiological and pharmacological factors.     

In vitro cultivation of Ostertagia ostertagi, the medium stomach worm of cattle. II. Effect of insect-growth-disrupting amines and amides on development

Thirteen secondary and tertiary amides and amines that disrupt growth and development in certain insects were tested at concentrations of 0.25–5.0 p.p.m. for their effects on development of Ostertagia ostertagi, from infective larvae to egg-laying adults, in a two-step roller culture system. These compounds did not affect exsheathment of infective larvae. However, some of these compounds inhibited development or killed 100% of the exsheathed larvae at concentrations of 1.0–2.5 p.p.m. Minimum inhibitive concentrations of several of the amines and amides affected the nematodes in one or more of the following ways: decreased motility or paralysis; reduced survival; delayed or blocked third or fourth ecdysis; lowered yields of advanced stages; decreased production of fertile and non-fertile eggs. The results also indicated that, as in insects, these chemicals exerted their lethal effects against O. ostertagi at the time of molt.     

Interrelationships among physicochemical properties, absorption and anthelmintic activities of 2-desoxoparaherquamide and selected analogs

The interrelationships between physicochemical properties, absorption and potency of 2-desoxoparaherquamide and five analogs, representing a new anthelmintic class, were evaluated in in vitro and in vivo assays. At pH 7.5, rates of drug absorption by the gastrointestinal nematode Haemonchus contortus and jird small intestine, parameterized by the permeability coefficient, P(e), ranged from 1.2-2.4 x 10(-4) cm/min (nematode) to 2.5-5.5 x 10(-3) cm/min (jird). In the jird intestine, absorption was pH-dependent, with P(e) at pH 7.5 being twice that at pH 4.5, reflecting the negative influence of protonation on transport of these weakly basic molecules. Each compound rapidly paralyzed H. contortus during in vitro exposure to therapeutically relevant concentrations (1-10 microm). The kinetics of drug action on motility in vivo mirrored their in vitro effects; motility concentrations were reduced in nematodes collected from jird stomach 3 h following oral drug dosing, by which time > or =50% clearance of the parasites had occurred. The nematode/medium partition coefficient K ranged from 10.1 to 16.1, consistent with the lipophilic nature of the compounds. The time required to reduce motility in vitro by 50% (t50*) and P(e) were used to determine C(n)*, the concentration of drug in the nematode at t50*, as an indicator of intrinsic potency. In the jird, the apparent potencies of the compounds were insensitive to route of administration (i.e. oral = i.v. = i.p. = i.m.) for H. contortus and two other gastrointestinal nematodes, Ostertagia ostertagi and Trichostrongylus colubriformis; topical administration, however, required three to 10-fold higher doses for equivalent efficacy.     

Inhibition of motility in isolated horse small intestine is mediated by κ but not µ opioid receptors

The effects of preferential μ (morphine), selective μ (fentanyl), selective κ (compound U69593) opioid receptor agonists, and nonselective (naloxone) and selective μ (naloxonazine) antagonists on equine small intestinal motility were evaluated in vitro. Samples of circular muscle from equine jejunum were placed in isolated organ baths and drug-induced modifications of both spontaneous and electrically evoked contractile activity were measured. None of the opioid agonists induced a significant change in spontaneous contractions. Fentanyl and U69593 reduced electrically induced contractions, whereas morphine reduced them only slightly. Naloxone competitively antagonised U69593, but both naloxone and naloxonazine were unable to counteract the inhibition of contractions induced by fentanyl. The inhibition of contractions shown by fentanyl is therefore probably not mediated by opioid receptors, but due to an anticholinergic activity of this drug. In summary, these data showed an inhibitory effect exerted by κ receptors on equine small intestinal motility, whereas the role of μ receptors seemed marginal and would need further characterisation.     

Activity of pulmonary intravascular macrophages in cats and dogs with and without adult Dirofilaria immitis

Pulmonary intravascular macrophages (PIMs), large (20-80 microm diameter) monocytes are present in sheep, pigs, and horses, but not in dogs, rats, rabbits, or primates. The present study evaluated the phagocytic activity of various organs in cats and dogs and determined the influence of Dirofilaria immitis infections on PIM activity. Live or dead adult heartworm (HW) was transplanted via jugular venotomy into cats and dogs. Cats (four per group) were allocated to five groups: surgical controls--no HW, dead HW for 1 week, live HW for 1 week, dead HW for 3 weeks, or live HW for 3 weeks. Radioactive technetium (Tc-99m, 1.2mCi in 0.3ml) sulfa-colloid was injected intravenously. All cats with HW were clinically asymptomatic and developed radiographic pulmonary parenchymal changes. No gross changes were visible at necropsy for cats with HW; inflammatory changes were less severe in cats with live HW. In cats with dead HW for 3 weeks, worms were present but folded, flattened, and located in distal pulmonary arteries. Uninfected control dogs and those with dead HW did not demonstrate any PIM activity. In control cats, lungs were the primary phagocytic organ after systemic IV colloid injection (72.5% of the total recovered radioactive dose). The lung and liver together represented over 95% of the recovered Tc-99m colloid in all cats. In each group of cats with HW, phagocytic activity of the lung was significantly less (p < 0.001) than the PIM activity of controls. Cats with dead HW at 1 week (50.1%) had a significant (p < 0.019) decrease in PIM activity compared with cats with dead HW at 3 weeks (59.5%). The PIM activity in cats with live HW was significantly decreased (p < 0.001) from that in groups with dead HW, but there was no significant difference between the two groups infected with live worms. There were no significant differences in recovery between any groups in pairwise analysis of the spleen, heart, skeletal muscle, kidney, bone marrow, or blood. Significant increases (p < 0.001) in liver activity for each group inversely reflected the decreased lung activity; consistent with increased hepatic uptake of Tc colloid "escaping" a relatively suppressed lung macrophage system. Transmission electron microscopy confirmed PIM glycocalyx changes and vacuolization, moderate Type 1 cell damage and Type II cell hypertrophy in cats with dead HW. There was no evidence of PIM death. The significant decrease in PIM activity in groups with dead HW and a greater decrease in groups with live HW are consistent with a down-regulation of PIM function in cats with live HW.     

Anthelmintic paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammals

Oxindole alkaloids in the paraherquamide/marcfortine family exhibit broad-spectrum anthelmintic activity that includes drug-resistant strains of nematodes. Paraherquamide (PHQ), 2-deoxoparaherquamide (2DPHQ), and close structural analogs of these compounds rapidly induce flaccid paralysis in parasitic nematodes in vitro, without affecting adenosine triphosphate (ATP) levels. The mechanism of action of this anthelmintic class was investigated using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum. None of the compounds altered A. suum muscle tension or membrane potential. However, PHQ blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by acetylcholine (ACh) and the nicotinic agonists levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic activity of PHQ and marcfortines is due to blockade of cholinergic neuromuscular transmission. The effects of these compounds were also examined on subtypes of human nicotinic ACh receptors expressed in mammalian cells with a Ca2+ flux assay. 2DPHQ blocked nicotinic stimulation of cells expressing alpha3 ganglionic (IC50 approximately 9 microm) and muscle-type (IC50 approximately 3 microm) nicotinic cholinergic receptors, but was inactive at 100 microm vs. the alpha7 CNS subtype. PHQ anthelmintics are nicotinic cholinergic antagonists in both nematodes and mammals, and this mechanism appears to underlie both their efficacy and toxicity.     

The uterine motility in cattle during late pregnancy, labor and puerperium. II. Drug modification

The uterine effects of oxytocin, the prostaglandins dinoprost and cloprostenol as well as clenbuterol, ergometrin, xylazine and Utrorale were investigated in 8 cows during late pregnancy, parturition and early puerperium (until 4th day p. p.). Uterine motility was measured by means of pressure microsensors and electrodes which were surgically implanted 3 to 4 weeks before parturition. Hysterograms were characterized by means of pressure amplitude, frequency and duration of uterine contractions and also by electromyography. Oxytocin (2-5 IE) given intravenously always provoked strong uterine contractions until the 4th day p.p. From the prostaglandins examined during early puerperium only dinoprost (15 mg i.v.) produced uterotonic effects, while the synthetic analogue cloprostenol (0.25 mg i.v.) had a weak stimulatory activity only on day 1 p.p. Both prostaglandins were ineffective when injected intramuscularly. Clenbuterol (0.3 mg i.v.) a beta 2-mimetic compound effectively induced long lasting tocolysis during parturition, which could be abolished by oxytocin. Xylazine (10 mg i.v.) was able to significantly increase uterine motility during late gestation. Following intravenous administration of ergometrin (1 and 10 mg), bunitrolol (1-16 mg) and Utrorale (0.1-4 ml) including its compounds oleum sabinae, oleum terebinthinae, balsamum copaivale and Styrax no uterokinetic activity was recorded at any time.     

变温条件下不同投喂水平对施氏鲟幼鱼消化酶、代谢酶和抗氧化酶活性的影响

本试验旨在研究变温条件下不同投喂水平对施氏鲟幼鱼消化酶、代谢酶和抗氧化酶活性的影响。试验水温设恒温[(22.0±0.1)℃]和变温[(22±2)℃]2种模式,并根据投喂水平分为6组,分别为恒温100%饱食组(HW-100组)、变温100%饱食组(BW-100组)、变温90%饱食组(BW-90组)、变温80%饱食组(BW-80组)、变温70%饱食组(BW-70组)、变温60%饱食组(BW-60组)。将平均体重为(34.9±0.8)g的施氏鲟幼鱼随机分为6组,每组4个重复,每个重复15尾鱼,养殖周期为42 d。结果表明:变温条件下,在60%~80%范围内,随着投喂水平的降低,胃及瓣肠蛋白酶活性均显著升高(P0.05)。与HW-100组相比,BW-60组与BW-70组的胃和瓣肠蛋白酶活性均显著升高(P0.05)。变温条件下,十二指肠和瓣肠淀粉酶活性及瓣肠脂肪酶活性均随投喂水平的降低呈先升高后下降的趋势。BW-80组十二指肠淀粉酶活性显著高于HW-100组及BW-100组(P0.05),BW-80组和BW-90组瓣肠淀粉酶活性显著高于其他各组(P0.05)。所有变温投喂组瓣肠脂肪酶活性均显著低于HW-100组(P0.05)。除BW-70组外,各变温投喂组血清丙氨酸转氨酶(ALT)活性均显著高于HW-100组(P0.05)。与HW-100组相比,BW-60组、BW-70组血清超氧化物歧化酶(SOD)活性显著降低(P0.05)。在变温条件下,在60%~90%范围内,鱼体血清总抗氧化能力(T-AOC)和黄嘌呤氧化酶(XOD)活性均随着投喂水平的降低整体呈先下降后上升的趋势,且均在投喂水平为80%时降至最低值。与HW-100组相比,BW-60组、BW-70组及BW-100组血清T-AOC显著升高(P0.05),且BW-60组和BW-90组血清XOD活性显著升高(P0.05)。综上所述,在周期性变温条件下,施氏鲟幼鱼可通过提高蛋白酶活性,即提高对饲料蛋白质消化效率的方式来补偿其对饲料蛋白质摄入的不足。与恒温100%饱食相比,变温条件下各投喂水平均可提高施氏鲟幼鱼血清ALT活性。     

Efficacy of avermectin B1 given orally against equine intestinal strongyles and Onchocera microfilaria

Three groups of horses and ponies (N = 13, 13 and 12) were treated with ivermectin paste (0.2 mg/kg p.o.), avermectin B1 solution (0.2 mg/kg p.o.), or fenbendazole suspension (10 mg/kg via nasogastric tube). The avermectin B1 was a 1% solution in a propylene glycolglycerol formal base. Faecal strongyle egg counts were performed before, and 14, 28, 42, 56 and 70 d, after treatment. Full-thickness skin biopsies from the neck, pectoral and umbilical regions were examined for Onchocera microfilaria before treatment, and again 14 and 70 d later. Ivermectin therapy produced a significant (P less than 0.01) decrease in mean strongyle egg counts 14, 28, 42 and 56 d after treatment. Avermectin B1 therapy resulted in significant (P less than 0.01) decreases in mean strongyle egg counts 14, 28 and 42 d after treatment. All horses given ivermectin or avermectin B1 had zero strongyle egg counts 14 and 28 d after treatment. Fenbendazole failed to significantly decrease strongyle egg counts. Both ivermectin and avermectin B1 resulted in zero microfilaria counts in all horses 14 d after treatment. On day 70 the percentage decrease in microfilaria counts were 100% and 99.6% respectively. Fenbendazole failed to significantly decrease microfilaria counts. The oral administration of this formulation of avermectin B1 appeared to be highly efficacious against intestinal strongyles and Onchocera microfilaria. The duration of anti-strongyle activity was, however, significantly (P less than 0.01) shorter than that of ivermectin paste.     

Screening of microfilaricidal effects of plant extracts against Dirofilaria immitis

Canine dirofilariasis is a common tropical parasitic disease of companion animals, caused by infestation of Dirofilaria immitis filarids within the pulmonary arteries and extending into the right heart. Increased reports of adverse reactions elicited by current microfilaricidal agents against D. immitis such as neurological disorders, circulatory collapse and potential resistance against these agents, warrant the search for new agents in forms of plant extracts. The use of plant extracts in therapeutic medicine is commonly met with scepticism by the veterinary community, thus the lack of focus on its medical potential. This study evaluated the presence of microfilaricidal activities of the aqueous extracts of Zingiber officinale, Andrographis paniculata and Tinospora crispa Miers on D. immitisin vitro at different concentrations; 10 mg/ml, 1 mg/ml, 100 μg/ml, 10 μg/ml and 1 μg/ml within 24 h, by evaluation of relative microfilarial motility as a measure of microfilaricidal activity. All extracts showed microfilaricidal activity with Z. officinale exhibiting the strongest activity overall, followed by A. paniculata and T. crispa Miers. It is speculated that the microfilaricidal mechanism exhibited by these extracts is via spastic paralysis based upon direct observation of the microfilarial motility.     

Antibabesial activity of protoberberine alkaloids and 20-hydroxyecdysone from Arcangelisia flava against Babesia gibsoni in culture

Bioassay-guided fractionation of the boiled extract from the stems of Arcangelisia flava led to the isolation of palmatine (1), berberine (2), jatrorrhizine (3), dihydroberberine (4) and 20-hydroxyecdysone (5). The chemical structures of these compounds were elucidated on the basis of their chemical and spectral evidence. The isolated compounds were evaluated for their growth inhibiting effects on Babesia gibsoni in culture for a week. Compounds (1-4) showed significant inhibitions at concentrations from 100 to 1.0 microg/ml, while compound 5 at a concentration of 100 microg/ml, only.     

Live weights at slaughter significantly affect the meat quality and flavor components of pork meat

One‐hundred‐twenty crossbred pigs finished at 175–185 days of age were used to investigate the effects of live weights at slaughter on the meat quality, volatile flavor compounds, and sensory attributes of pork meat. Based on the live weights at slaughter, three weight groups (n = 36 per group) were classified as follows: light weight (LW: 100 kg), medium weight (MW: 110 kg), and heavy weight (HW: 120 kg). After slaughter, longissimus dorsi muscle samples were taken and used for the analyses of aforementioned parameters. The HW group had higher fat content and water holding capacity compared to the LW or MW group (p < 0.05). The HW group also showed higher levels for majority of unsaturated fatty acids and total polyunsaturated fatty acids than the LW group (p < 0.05). The slaughter weight significantly affected the amounts of 11 among the 47 flavor compounds identified. Significantly higher amounts of fatty acids oxidation‐derived flavor compounds (aldehydes) were found in the HW group than in the other groups. Noticeably, increasing slaughter weight was associated with higher sensorial scores for flavor, juiciness, and acceptance scores (p < 0.05). Based on the obtained results, 120 kg body weight is recommended as the market weight for this commercial breed without compromising the meat quality.     

Effects of dietary concentrations of methionine on growth performance and oxidative status of broiler chickens with different hatching weight

1. A study was conducted to evaluate the effects of two hatching weight (HW) levels and two dietary concentrations of methionine on the growth performance and oxidative status of broilers. Male Arbor Acres chickens were divided into two groups on their HW (low and high HW, H and L). Each HW group was then distributed into two subgroups, of similar HW, receiving either low or high dietary concentrations of methionine (4.9 g methionine/kg, LM; 5.9 g methionine/kg, HM). Thus, all day-old birds were distributed into 4 treatments (H-LM, H-HM, L-LM, L-HM) × 6 replicates × 10 birds for 21 d.

2. Broilers with high HW were heavier than those with low HW during the 21 d assay, which appeared to result from increased body weight gain rather than improved feed conversion efficiency. A higher dietary concentration of methionine (5.9 g/kg) improved growth performance of broilers with low HW in terms of body weight gain and feed conversion ratio.

3. Broilers with different HW had similar antioxidant status both in serum and liver.

4. Broilers given a diet containing 5.9 g/kg methionine had enhanced serum superoxide dismutase (SOD) activity and decreased hepatic malondialdehyde (MDA) content at day 7.

5. Broilers given a diet containing 5.9 g/kg methionine had a higher hepatic reduced glutathione (GSH):glutathione disulphide (GSSG) ratio than those given a diet containing 4.9 g/kg methionine at day 21. High dietary methionine concentration reduced hepatic GSH content and glutathione peroxidase (GPX) activity of broilers with high HW at day 7 and at day 21, respectively, but increased hepatic GSH content of broilers with low HW at day 7.

6. Although broilers with different HW had similar oxidative status as indicated by several parameters in blood and liver, HW can have positive effects on the subsequent growth performance of broilers, and a higher dietary methionine concentration (5.9 g/kg) can improve growth performance and antioxidant status in broilers exhibiting low HW.     

Nematocidal activities of thiabendazole and ivermectin against the larvae of Strongyloides ratti and S. venezuelensis

With the aim of developing therapeutic agents for strongyloidosis, the disease caused by infection with Strongyloides stercoralis, we established a novel assay technique using S. ratti and S. venezuelensis as models for S. stercoralis. The newly developed assay technique was found to more accurately represent treatment-induced larval paralysis than existing assays. Our method uses paper disks impregnated with the test solution, which even allows materials that are sparingly soluble in water to be tested. An inverted microscope was used to observe the larval states, and these states were recorded using a digital camera. We observed the activities of ivermectin and thiabendazole against larvae and calculated larval motility and velocity. These two factors were then combined to determine the overall viability of larvae at selected concentrations. The activities of the anthelmintics were compared by calculating the concentrations at which 50% viability was demonstrated, or in other words, the concentration at which paralysis was caused in 50% of the individuals (50% paralysis concentration; PC(50)). Evaluations after 24h of exposure yielded the following reproducible PC(50) values for ivermectin and thiabendazole, respectively: S. ratti, 2.4 and 140 microM; and S. venezuelensis, 2.3 and 190 microM. After treatment with ivermectin, there was a tendency for larval motility to be greater than that of the controls at low concentrations, a result that might be associated with its mechanism of action.     

Screen for anthelmintics, using larvae of Ascaris suum

A multiwell culture system was used to assay the effects of 12 known anthelmintic compounds on Ascaris suum larval development from 2nd-stage (L2; hatched from eggs) to early 3rd-stage (L3) and from in vivo-derived late L3 to early 4th-stage (L4). Larval survival, development, and motility were monitored for drug effects. Development of L2 to L3 was sensitive to thiabendazole, albendazole (ABZ), ABZ/sulfoxide, ABZ/sulfone (SO), mebendazole, L-tetramisole, D-tetramisole, piperazine, or closantel at a concentration of 0.01 microgram/ml; however, the effects of these drugs on larval development did not correlate well with known effects in vivo. The development of L3 to L4 was blocked by ABZ or mebendazole at 0.01 microgram/ml, by thiabendazole or ABZ/sulfoxide at 0.1 microgram/ml, and by ABZ/SO at 1.0 microgram/ml; however, except for ABZ/SO, most larvae were viable at these concentrations. In contrast, L-tetramisole or morantel appeared to inhibit development of L3 to L4 and to reduce survival at concentrations of greater than or equal to 1 microgram/ml; however, D-tetramisole was at least 10 times less effective. Haloxon, ivermectin, and closantel blocked development of L3 to L4 at 0.1, 1, and 10 micrograms/ml, respectively, in the absence of serum, but their activity was reduced by the presence of serum. Seemingly, in vitro development of A suum larvae was a convenient and sensitive bioassay for anthelmintic activity and could serve as a screen for anthelmintic residues in edible tissues.     

Single nucleotide polymorphisms in several porcine cathepsin genes are associated with growth, carcass, and production traits in Italian Large White pigs

To identify DNA markers associated with performance, carcass, and meat production traits including muscle postmortem cathepsin activity, several porcine genes encoding for lysosomal proteinases (cathepsin B, CTSB; cathepsin D, CTSD; cathepsin F, CTSF; cathepsin H, CTSH; cathepsin L, CTSL; and cathepsin Z, CTSZ) and for a cathepsin inhibitor (cystatin B) were investigated. Single nucleotide polymorphisms were identified in CTSD, CTSH, CTSL, and CTSZ genes with a combination of in silico expressed sequence tag database mining and single-strand conformation polymorphism analysis. Sequencing and PCR-RFLP protocols were used to validate the identified polymorphisms. Allele frequencies at these loci were investigated in Italian Large White, Landrace, Duroc, Piétrain, Belgian Landrace, Hampshire, and Meishan breeds. Genotyping CTSD and CTSH markers made it possible to genetically map these genes to SSC 2 and 7, respectively. Markers in CTSD, CTSH, CTSL, and CTSZ genes, together with mutations we previously reported in cystatin B, CTSB, and CTSF genes, were genotyped in an Italian Large White sib-tested population (272 or 482 animals). For these animals, meat quality traits (cathepsin B activity, pH measured at 2 h postmortem, pH measured at 24 h postmortem, glycogen, lactate, and glycolytic potential of semimembranosus muscle) and EBV for ADG, lean cuts (LC), backfat thickness (BFT), ham weight (HW), and feed:gain ratio (FGR) were determined. Analyzed markers did not show any association with muscle cathepsin B activity. Thus, it could be possible that different genes, other than these investigated candidates, affect this trait, which is correlated with the excessive softness defect of dry-cured hams. The results of association analysis confirmed the effects we already reported in another study for CTSF on ADG (P = 0.008), LC (P = 0.001), and BFT (P = 0.02). Moreover, CTSD was associated with ADG, LC (P < 0.0001), BFT, HW, and FGR (P < 0.001); CTSH was associated with FGR (P = 0.026); and CTSZ was associated with ADG (P = 0.006), LC (P = 0.01), HW (P = 0.024), and FGR (P = 0.029). The biochemical and physiological functions of the lysosomal proteinases, together with the results obtained in our investigation, suggest that the cathepsin gene family might play important roles affecting economic traits in pigs.     

Chemotactic and chemokinetic activity of Streptococcus faecalis culture supernatant for equine neutrophils

Although equine neutrophils did not respond towards formylated methionyl peptides, Streptococcus faecalis culture supernatant caused an in vitro stimulation of equine neutrophil motility when measured by an under-agarose assay. The migration of neutrophils towards the culture supernatant increased sigmoidally with the logarithmic concentration of the culture supernatant in the chemoattractant wells. The streptococcal culture supernatant was chemokinetic because it stimulated the random motility of the phagocytes. Because granulocytes migrated further towards the supernatant than could be explained by the chemokinetic activity of the bacterial products, the streptococcal culture fluid also exerted a chemotactic effect on the leukocytes. The chemotactic activity of the supernatant was further confirmed by the changes in the orientation of the migrating cells during incubation. These results indicate that bacteria produce cytotaxins other than formylmethionyl peptides which are recognized by equine neutrophils.