Contractile activity and motility responses of the dog heartworm Dirofilaria immitis to classical anthelmintics and other compounds

A variety of compounds including classical anthelmintics and avermectin analogs were screened for their effects on movements of adult heartworms (HW) (Dirofilaria immitis). Contractile activity was measured by tension recording of spontaneous movements of intact HW coil preparations (6 min compound exposure) and motility was evaluated by observation of spontaneous, free movements in culture (3 and 7 days compound exposure). Results for female HW indicated that some compounds caused spastic paralysis of contractile activity and inhibition of motility in culture (bephenium, DL-tetramisole, and pyrantel); some caused only spastic paralysis of contractile activity (methyridine and disophenol); and some caused only inhibition of motility in culture (chlorpromazine, dithiazanine, 1-ethoxycarbonylmethyl-1-methylpyrrolidinium, and 4-methyltropolone). Effects on motility in culture appeared to be lethal. The following compounds lacked effects: amprolium, 2-amino-2-thiazoline, bithionol, bitoscanate, bitriben, hexachlorophene, ivermectin, and 10 H-phenothiazine. A group of avermectin analogs was screened for effects only on motility in culture of both adult female and male HW. Several of the analogs affected motility, but the effects appeared to be non-lethal. Microfilaria release into the culture media was suppressed by two of the analogs (an aglycone and avermectin B2). The HW maintenance system used in the present study facilitated screening of compounds for effects on this parasite.     

Larval paralysis as an in vitro assay of levamisole and morantel tartrate resistance inOstertagia

The paper describes a method for detecting levamisole and morantel tartrate resistance by determining the percentage of paralysed third stage larvae in serial dilutions of anthelmintic. Levamisole resistantOstertagia spp. had a smaller proportion of larvae undergoing tonic paralysis in either levamisole or morantel tartrate than did a non-resistant strain. The dose response thus obtained for a strain of worms can be tested statistically against strains which are known to be resistant or non-resistant.Commonwealth of Australia     

Interactions between erythromycin,flunixin meglumine,levamisole and plant secondary metabolites towards bovine gastrointestinal motility—in vitro study

Continued ingestion of plant secondary metabolites by ruminants can provoke pharmacological interactions with pharmaceutical agents used in animals. As some drugs and phytocompounds affect smooth muscle activity, the aim of this study was to verify the possible interaction between selected pharmaceutical agents and plant secondary metabolites towards bovine gastrointestinal motility. The interactions between phytocompounds—apigenin, quercetin, hederagenin, medicagenic acid—and medicines—erythromycin, flunixin meglumine and levamisole—were evaluated on bovine isolated abomasal and duodenal specimens obtained from routinely slaughtered cows. The obtained results confirmed the contractile effect of all three drugs used solely. Hederagenin and medicagenic acid (0.001 μM) enhanced the contractile effect of levamisole. Hederagenin additionally increased the impact of erythromycin. Both saponins (100 μM) showed synergistic effects with all tested pharmaceuticals. Apigenin and quercetin (0.001 μM) intensified the contractile response induced by erythromycin and levamisole. Moreover, both flavonoids (100 μM) showed an antagonistic interaction with all tested drugs which in that situation were devoid of the prokinetic effect. To conclude, plant metabolic metabolites such as saponins and flavonoids are potent modifiers of the effect of drugs towards gut motility. The synergy observed between phytocompounds and selected medicines can be beneficial in the treatment of cows with hypomotility disorders.     

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.     

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.     

Suspected tick paralysis (Ixodes holocyclus) in a Miniature Horse

A 9‐year‐old Miniature Horse gelding infested with ticks (Ixodes holocyclus) was presented with flaccid motor paralysis causing recumbency. Neurological examination and other diagnostic tests did not identify an alternative aetiology, leading to a presumptive diagnosis of tick paralysis. The gelding was treated with tick antiserum and intensive supportive care. He made a gradual recovery over the 48 h following presentation and was discharged without further complications. This case report describes in detail the clinical signs and successful treatment of a Miniature Horse with flaccid paralysis caused by suspected envenomation by Ixodes holocyclus.     

The effect of levamisole and albendazole on some enzymes of Ascaridia galli and Heterakis gallinae

Ascaridia galli and Heterakis gallinae obtained from the common fowl Gallus gallus were exposed to 10(-2)-10(-5)M levamisole and albendazole; both compounds caused death of the parasites in vitro. The effect of the drugs was investigated on homogenates of the treated worms. Albendazole, at 10(-2)M, inhibited oxaloacetate reduction by 67 and 53% and malate oxidation by 21 and 17% in A. galli and H. gallinae, respectively, whereas 10(-4)M levamisole completely inhibited malate dehydrogenase activity in both directions in the two parasites. Lactate dehydrogenase was not affected significantly by either anthelmintic. Aldolase activity was diminished by 57 and 32% in A. galli and H. gallinae, respectively, with 10(-4)M levamisole. Levamisole at 10(-4)M also inhibited the activity of acid and alkaline phosphomonoesterase and cholinesterase. Albendazole had no significant effect on these enzymes in either parasite. Malate dehydrogenase and cholinesterase activity of the host tissue (intestine and caecum) was also reduced significantly with 10(-2) and 10(-3)M levamisole. These studies indicated a multiple mode of action of levamisole and albendazole.     

Evolution of High-level,Multiple Anthelmintic Resistance on a Sheep Farm in Malaysia

Anthelmintic resistance in nematode parasites of sheep and goats on a government farm in north Malaysia was monitored over a 3-year period (1997–2000). The faecal egg count reduction test (FECRT) was conducted on young sheep at the beginning and end of this period. Changes in management, designed to reduce the selection pressure for the development of anthelmintic resistance, were also implemented during this time. By far the most important parasite problem was Haemonchus contortus. In 1997, this nematode was found to be resistant to levamisole, with suspected resistance to closantel and moxidectin. However, when the FECRT was repeated 3 years later, its resistance status had become much more severe, with resistance to benzimidazole, levamisole and ivermectin, and suspected resistance to moxidectin. This rapid evolution to multiple anthelmintic resistance is a major concern that needs to be arrested. There is an urgent need to evaluate other control strategies that incorporate livestock management, the `smart' use of drugs and non-chemotherapeutic approaches, such as biological control agents.     

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.     

In vitro lecithinase activity and sensitivity to 22 antimicrobial agents of Clostridium perfringens isolated from necrotic enteritis of broiler chickens

Viable Clostridium perfringens ranging from 10(5) to 10(8) g-1 was detected in all of 88 intestinal content specimens of necrotic enteritis in broiler chickens. In vitro lecithinase activity and sensitivity to 22 antimicrobial agents were determined for the 88 isolates. The activities of lecithinase in the culture filtrate of isolates were estimated to be 0.5 to 4.0 AE ml-1 as alpha-antitoxin equivalent. With reference to antimicrobial activity penicillins and cephazolin showed excellent activity and no resistance; peptides, of the agents used as growth promoters, showed that all except bacitracin had low minimum inhibitory concentration levels (1.6 micrograms ml-1 or less) against this organism; polyethers of monensin, salinomycin and lasalocid were generally adequate in low concentrations while there was a high level of resistance to three tetracyclines in 90 per cent of the strains and all isolates were insusceptible to streptomycin of the aminoglycoside antibiotics.     

Comparison of two versions of larval development test to detect anthelmintic resistance in Haemonchus contortus

Larval development (LDT) and micro-agar larval development tests (MALDT) were used to compare the reliability and sensitivity of two methods for detecting anthelmintic resistance in Haemonchus contortus. The tests were conducted using three resistant and four susceptible isolates of H. contortus. Both versions of the tests provided comparable results with regard to the characterization of benzimidazole and levamisole susceptibility but neither test was sufficiently sensitive to discrimination between an ivermectin (IVM) susceptible and an IVM resistant isolate. Each test has its own merits with the LDT having the advantage of being less time-consuming.     

Synergetic effects of oral administration of levamisole and Echinacea purpurea on immune response in Wistar rat

This research investigated the effects of levamisole and Echinacea purpurea (EP), separately and together on the immune response of the rat as a laboratory model. In this experiment, 40 male Wistar rats were obtained and divided into four groups (n = 10). These groups received normal saline (10 mg/kg), EP (500 mg/kg), levamisole (2 mg/kg) and EP (500 mg/kg) with levamisole (2 mg/kg) as oral gavages every day for a period of 4 weeks, respectively. After obtaining blood samples (at the end of each week), haematocrit (HCT), differential and total white blood cell (WBC) counts, phagocyte activity (number), total protein, albumin and globulins levels of samples were obtained. The results of the study showed that the gamma globulin level, WBC, neutrophil and monocyte counts and phagocyte activity increased significantly in comparison with normal saline group during the study. According to the results, each of the mentioned agents had a stimulant effect on the immune system separately and together on rats. In the group that received EP and levamisole simultaneously, these effects were synergistically increased. These compounds, by increasing the mentioned factors, will probably affect the immune system.     

Reliability and reproducibility of the larval paralysis test as an in vitro method for the detection of anthelmintic resistance of nematodes against levamisole and morantel tartrate

In order to study the reliability of the larval paralysis test as an in vitro assay for the detection of resistance of nematodes to levamisole and morantel tartrate, the influence of different parameters was evaluated using resistant and susceptible Ostertagia ostertagi strains. The operator, the sample (10% of the larvae present in the suspension), the incubation time (24, 48 or 72 h), the incubation temperature (20 or 25 degrees C) and the observation period of the larvae (5 or 15 s) had no statistically significant influence on the test results. Statistical differences were obtained only when L3 larvae of different ages (1 or 2 months) were used. Reversibility of the paralysis did not occur when concentrations of levamisole of less than or equal to 200 micrograms ml-1 or morantel tartrate of less than 2000 micrograms ml-1 were used. The reproducibility of the test was fairly good, with a mean standard deviation of 21.3% for the LC50 values. Morantel resistance in a strain of O. ostertagi was not confirmed as such by the larval paralysis test. The resistance factor was less than or equal to 1, in spite of an efficacy of morantel of 77.4% as shown in vivo.     

Clinical application of prokinetics.

Intestinal stasis or ileus is a significant cause of mortality and morbidity in horses and has been attributed to a variety of causes, including loss of intrinsic or extrinsic electrical activity, incoordination of contractile activity from regional stimuli, and dissociation between electrical and mechanical activity. Proposed mechanisms include systemic shock, electrolyte disturbances, persistent luminal distention, ischemia, inflammation, peritonitis, endotoxemia, and anesthesia. Because the cause of ileus is likely multifactorial, a variety of pharmaceutics have been used to target specific causes. Prokinetics are defined as agents that facilitate or enhance the net movement of feed material down the length of the intestinal tract and do not simply produce an uncoordinated increase in local contractile activity. The primary objective of pharmaceutic intervention is to augment the pathways that stimulate motility or attenuate the inhibitory neurons that predominantly suppress activity. The objective of this article is to summarize the actions of prokinetic agents available and suggest clinical applications.     

Role of Endothelium and Nitric Oxide in the Response of Equine Colonic Arterial Rings to Vasoconstrictor Agents

Objective- To determine the in vitro contractile responses of equine colonic arteries to angiotensin II, histamine, serotonin, norepinephrine, prostaglandin F_2α, vasopressin, and a thromboxane-B₂-analogue. Study Design- The tension generated in colonic arterial rings placed in organ baths with oxygenated Tyrode's solution at 37°C after exposure to the previously mentioned chemical agents was measured using force-transducers interfaced with a polygraph. Sample Population- Large colon arterial rings collected from eight horses. Methods- The rings were allowed to equilibrate for 45 minutes after applying 2 g tension. Bath solution was replaced and tension reapplied at 15-minute intervals. Cumulative-concentration-responses were determined for concentrations ranging from 10^-8M to 10^-4M on three vessel groups namely endothelium intact, endothelium denuded, and L-NAME treated. The maximal response for each vessel was considered as 100%; responses to lower concentrations were calculated as a percentage of the maximum. The EC₅₀ value was determined for each concentration-response relationship of each agent. Results- Vessels with denuded endothelium or those incubated with L-NAME had greater contractile responses. Angiotensin, histamine, serotonin, and norepinephrine produced greater maximal responses than the other agents. Endothelium denuded rings had lower EC₅₀ values. Responses to norepinephrine and serotonin were affected less by denudation. Conclusion- Endothelium plays an important role in modulating responses of colonic arterial rings to contractile agents. Endothelium-derived vasodilators, other than nitric oxide, may modulate contractile responses of equine colonic arteries. Clinical Relevance- Endothelial damage associated with colonic vovulus may be a major factor for sustained reduced perfusion after surgical correction.     

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoró, Rio Grande do Norte, Brazil

Toxoplasma gondii is an obligate intracellular parasite with a variety of hosts, responsible for reproductive problems and economic losses in sheep flocks. Neospora caninum was recently identified and its clinical presentation in sheep is similar to that of toxoplasmosis, which can cause repeated abortions, though less frequently in this species. In order to confirm the prevalence of these agents in the city of Mossoró, Rio Grande do Norte, Brazil, 409 serum samples from adult sheep (364 females and 45 males) were tested by the indirect immunofluorescence antibody test, using cut-off point at a dilution of 1:64 and 1:50 for T. gondii and N. caninum, respectively. From the 35 properties examined, 23 (65.7%) had at least one seropositive animal for T. gondii and six (17.1%) for N. caninum. The prevalence of seropositive animals for T. gondii was 20.7% and for N. caninum 1.8%. There was no association between the presence of the agent’s antibody and gender, reports of reproductive problems and presence of dogs and/or cats in the properties. T. gondii is well distributed and N. caninum has low prevalence in sheep and in the properties of the studied region.     

Efficacy of Albendazole and Levamisole against Gastrointestinal Nematodes of Sheep and Goats in Morogoro,Tanzania

A study was conducted to determine the efficacy of albendazole after it had been withdrawn from use due to the development of resistant strains of nematodes about ten years ago. The study also aimed to determine the present efficacy of levamisole, which had been recommended to replace albendazole. On one farm, the sheep and goats were divided into two groups, one group of each serving as the untreated control, while the other was treated with levamisole. The sheep on the other farm were divided into three groups, one serving as the untreated control group, the second being treated with levamisole and the third being treated with albendazole. Faecal samples were collected one day before treatment, and again 10 days after treatment. Anthelmintic efficacy was determined by the faecal egg count reduction test. Ten days after treatment, the sheep treated with levamisole on the first farm had a 98% reduction in faecal egg count, with a 95% confidence limit of 76%. The goats on the same farm had a 97% reduction in faecal egg count, with a 95% lower confidence limit of 81%. At the second farm, 10 days after treatment, sheep treated with levamisole had a 99.4% reduction in faecal egg count, with a 95% lower confidence limit of 88.9%, whereas the sheep treated with albendazole only had a 59.4% reduction in faecal egg count, with a 95% lower confidence limit of –19.6%. The study indicated that the gastrointestinal nematodes of sheep at the Department of Animal Science and Production farm were still resistant to albendazole about ten years after this anthelmintic had been withdrawn from use. A reduced efficacy of levamisole was suspected.     

Field studies investigating anthelmintic resistance in young cattle on five farms in New Zealand

AIM: To investigate the efficacy of pour-on anthelmintics against field strains of parasitic nematodes in young cattle on five farms in New Zealand.

METHODS: Faecal nematode egg count (FEC) reduction (FECR) tests were carried out on five calf-rearing farms using pour-on formulations of levamisole, ivermectin, eprinomectin, and the simultaneous administration of levamisole and ivermec- tin. Faecal samples were collected per rectum before treatment and about 7, 14, 21 and 28 days after treatment, for FEC and faecal nematode larval culture.

RESULTS: Resistance (i.e. <95% reduction in FEC) of Cooperia oncophora to ivermectin and eprinomectin was identified on all five farms. There was limited evidence of possible emerging resistance in Ostertagia spp to ivermectin but not eprinomectin, in short-tailed larvae of Cooperia spp to ivermectin and eprinomec- tin, and in Trichostrongylus spp to ivermectin, eprinomectin and levamisole used separately. Levamisole was effective against C. oncophora, but had variable efficacy against Ostertagia spp in the calves in this study. Simultaneous treatment with levamisole and ivermectin pour-on formulations were effective against all genera on all farms.

CONCLUSIONS: To effectively manage roundworm parasites in their calves farmers need to be aware of the resistance status of the parasites on their farms. Levamisole is likely to be an effective anthelmintic on most farms at times of the year when the impact of Ostertagia spp is not high. Simultaneous administration of levamisole and ivermectin pour-on anthelmintics to cattle is likely to control both ML-resistant C. oncophora and stages of Ostertagia spp that are not controlled by levamisole alone.     

In vitro effects of nonsteroidal anti-inflammatory agents and prostaglandins I2, E2, and F2alpha on contractility of taenia of the large colon of horses.

OBJECTIVES: To determine the in vitro effect of various prostaglandins (PG) and nonsteroidal anti-inflammatory drugs (NSAID) on contractile activity of the large-colon taenia of horses. ANIMALS: 14 healthy horses. PROCEDURE: The taenia was collected from the ventral colon, cut into strips (2 X 10 mm), and mounted in a tissue bath system (20-ml capacity) that contained oxygenated Krebs buffer solution warmed to 37.5+/-0.5 C. After equilibration, incremental doses of PGE2, PGF2alpha, PGl2, flunixin meglumine, carprofen, ketoprofen, and phenylbutazone were added to the baths, and contractile activity was recorded. Magnitude of the response was calculated by comparing contractile activity before and after administration of the PG or NSAID to the tissue baths. RESULTS: PGE2 and PGF2alpha, caused a significant increase in contractile activity, whereas PGI2 induced an inhibitory response. Activity of NSAID on contraction was predominantly inhibitory. At low concentrations, ketoprofen induced an excitatory effect, which then became inhibitory at high concentrations. Compared with the other NSAID, carprofen significantly reduced contractile activity at lower concentrations. CONCLUSIONS: PGE2 and PGF2alpha appear to enhance contractility of large-colon taenia of horses, whereas PGI2 was inhibitory in the in vitro model. Administration of NSAID also inhibited contractility, with carprofen having the most potent effect. CLINICAL RELEVANCE: Administration of NSAID in combination with liberation of endogenous PG may predispose horses to development of intestinal stasis and subsequent impaction.     

Motility response of levamisole/benzimidazole-resistant Haemonchus contortus larvae

Electronic measurements of the motility of third stage larvae of a susceptible and a levamisole/benzimidazole-resistant strain of Haemonchus contortus were made after incubation in solutions of anthelmintics for 24 h. Results confirmed the resistance to benzimidazoles, but failed to show differences in response to levamisole. Visual observations of paralysis of first and third stage larvae in solutions of levamisole also failed to show significant differences between the two strains. The tests as conducted failed to demonstrate levamisole resistance, suggesting that nematode strains may vary in their mechanisms of resistance to levamisole.