Population replacement of benzimidazole-resistant Haemonchus contortus with susceptible strains: evidence of changes in the resistance status.

The spread of anthelmintic resistance (AR) in nematode populations threatens the viability of sheep production systems worldwide, and warrants the adoption of sensitive, practical, and standardized tests to detect AR. The aim of this study was to characterize the replacement of an Haemonchus contortus population resistant to benzimidazoles (BZDs) by a susceptible one, by means of both phenotypic and genotypic techniques. Phenotypic methods to assess BZD resistance included in vivo tests, such as the fecal egg count reduction test (FECRT), and in vitro tests, such as the egg hatch assay (EHA). Additionally, genotypification of polymorphisms associated with BZD resistance by sequencing a fragment of the isotype 1 ß-tubulin gene was carried out. The initial, BZD-resistant population (initial Balcarce population) exhibited an egg count reduction (ECR) of 59.3%. Following refugium replacement, the final population (final Balcarce population) exhibited an ECR of 95.2%. For the initial Balcarce population, the median effective dose (ED50) for the EHA was 0.607 µg thiabendazole (TBZ)/mL, with a rate of eclosion at a discriminating dose (EDD) of 0.1 µg TBZ/mL of 76.73%. For the final Balcarce population, ED50 was 0.02 µg TBZ/mL, and EDD was 1.97%. In the initial population, 93% of the analyzed individuals exhibited genotypic combinations associated with BZD resistance (53% Phe/Phe167-Tyr/Tyr200, 37% Phe/Tyr167-Phe/Tyr200, and 3% Phe/Tyr167-Glu/Leu198). Conversely, no combination associated with resistance was found in individuals from the final population. All of the tests were useful for detecting AR to BZDs. The results from the genetic and phenotypical studies were consistent, and the resulting information greatly aided in interpreting the outcomes of the population replacement and the potential impact of this strategy on management of AR.

Influence of faecal culture media and incubation time on the yield of infective larvae of Haemonchus contortus (Rudolphi 1803).

The aim of this experiment was to determine the yield of Haemonchus contortus third-stage larvae (L3) in faecal cultures in different conditions, including incubation time (7 or 14 days), the addition of inert additives (polystyrene pellets, vermiculite or no additive) and physical condition of the incubated faeces (ground or whole pellets). Twelve groups of 10 cultures each were arranged and incubated at 24 °C to evaluate the interaction of the above-mentioned conditions. Significantly, more L3 (p=0.0019 to p=0.0200) were recovered from cultures incubated for 7 days than for 14 days, except for the groups containing whole pellets with no additives (p=0.53) or with vermiculite (p=0.41). Larval yields from 7-day incubated cultures did not differ between groups (p=0.47), but for the whole pellets with vermiculite group, which yielded significantly less L3 (p<0.0001) than the rest of the cultures. Incubation for 14 days showed that cultures containing whole pellets with no additives yielded significantly more L3 (p<0.05) than the rest. Culturing faeces with H. contortus seems not to require inert additives or extra manipulation to obtain good L3 yields.

Recovery of fenbendazole efficacy on resistant Haemonchus contortus by management of parasite refugia and population replacement.

The recovery of fenbendazole efficacy against Haemonchus contortus was attempted in a sheep intensive production system, using a strategy of population replacement in which the initial absolute efficacy of fenbendazole was 0%. The strategy was based on managing the parasite populations in refugia. Firstly, the resistant parasite population was reduced by means of anthelmintic treatments with efficacious drugs (Phase I), then a new, susceptible population was introduced in summer by way of artificially infected lambs at weaning, which were left to graze on the experimental pasture for eleven months (Phase II). Lastly, the impact of the replacement strategy, in terms of benzimidazole efficacy, was measured (Phase III). Faecal egg counts from permanent lambs and worm burdens as a measure of pasture infectivity from tracer lambs were determined throughout the study. During Phase I, faecal egg counts diminished from a peak of 2968 (300-7740) epg to 0 epg at the end, while adult worm burdens of H. contortus were reduced from 2625 (800-5100) to 0, which showed that the treatment strategy used in Phase I was effective in reducing the resistant population. These parameters also showed that good levels of pasture contamination and infectivity were achieved in Phase II, as faecal egg counts of up to 7275 (3240-13080) epg and adult worm burdens of 500 (200-800) H. contortus were reached. The absolute benzimidazole efficacy on H. contortus estimated at 16 months post-population replacement (Phase III) was 97.58%. The results lead to the conclusion that the recovery of anthelmintic efficacy of fenbendazole against a resistant population of H. contortus may be achieved by means of a strategy based on management of refugia and a subsequent introduction of a susceptible population. This strategy might be translatable to other resistant nematode genera.

Anthelmintic resistance: Management of parasite refugia for Haemonchus contortus through the replacement of resistant with susceptible populations.

Sheep production in tropical and temperate regions is hampered by the presence of Haemonchus contortus, the blood-sucking nematode that is the major cause of economic losses in small ruminant enterprises. The most limiting factor in the control of this parasitic disease is the steady progress of anthelmintic resistance worldwide. The search for control strategies that minimise the use of anthelmintics is therefore central to various efforts worldwide. One strategy is the introduction of susceptible parasites in refugia when these refugia are at low levels. This strategy could lead to a renewed possibility anthelmintics being effective. At farm level, this management practice could recover the use of anthelmintics in flocks with high levels of resistance. This review explores the possibility of replacing resistant H. contortus populations with susceptible ones through refugia management and. highlights the experiences of on-farm research attempts carried out in different geographical areas, reaching various degrees of success.

Studies on vertical transmission of Trichinella spiralis in experimentally infected guinea pigs (Cavia porcellus).

An experimental study to enhance knowledge on the capability of Trichenella spiralis to pass from guinea pigs to progeny at different periods of pregnancy or lactation was performed. For this purpose, 18 female adult guinea pigs were inoculated with 100 or 1000 T. spiralis muscle larvae (ML) during early, late gestation and during lactation period. The presence of T. spiralis (ML) in mothers and newborns was studied through enzymatic digestion from muscle samples. ML were observed in 9 of 42 newborn guinea pigs and levels of infection were significantly higher when infections of mothers were done during late gestation (p = 0.0046) with the high infective dose (p = 0.0043). T. spiralis ML were not recovered from any of the newborns from mothers infected in the lactation period. Ten out of 18 infected mothers presented larvae 1 in their mammary glands. Muscle samples from the tongue and the masseter showed the highest larval burdens. These observations confirm previous reports on that ML of T. spiralis are capable to pass through placental tissues to reach and encyst in striated muscle groups of newborn guinea pigs. This study may also reinforce the importance of preventive programs to control trichinellosis in those endemic areas where pregnant women would have high risk of infection.

Resistant nematodes in cattle: Pharmaco-therapeutic assessment of the ivermectin- ricobendazole combination.

Nematodicidal combinations have been proposed as a valid strategy to achieve effective nematode control in the presence of drug resistance. The goals of this study were: (1) to compare the clinical efficacy (therapeutic response) of ivermectin (IVM) and ricobendazole (RBZ) given subcutaneously either by separate or combined administration to calves naturally infected with gastrointestinal nematodes resistant to IVM, and (2) to evaluate the potential pharmacokinetic (PK) and/or pharmacodynamic (PD) interactions occurring after the co-administration of both anthelmintics. Sixty male calves naturally infected with gastrointestinal nematodes resistant to IVM were randomly allocated into four groups (n=15). Untreated control: animals not receiving anthelmintic treatment; IVM alone: animals treated with IVM by subcutaneous (SC) injection (0.2mg/kg); RBZ alone: animals received RBZ by the SC route (3.75mg/kg); IVM+RBZ: animals treated with IVM and RBZ (0.2 and 3.75mg/kg, respectively), by SC injection in two separates sites. Eight animals of each treated group were randomly selected to perform the PK study. Plasma samples were taken from those animals up to 28days post-treatment. IVM and RBZ plasma concentrations were quantified by HPLC. The therapeutic response was determined by faecal egg count reduction test (FECRT). The proportions of third-stage larvae (L3) recovered from coprocultures were used to calculate the efficacy against the main parasite genera. The daily total egg deposition for each experimental group was estimated. Similar pharmacokinetic trends were obtained for both IVM and RBZ allying the single-drug and the combined treatments, which indicates the absence of PK interactions between both anthelmintics. The observed overall clinical drug efficacies were 48% (IVM alone), 94% (RBZ alone) and 98% (IVM+RBZ). Haemonchus spp. and Cooperia spp. were recovered in the coproculture after IVM treatment, suggesting that resistance to IVM includes both genera. In fact, the efficacy against Cooperia spp. was 83% (IVM), 98% (RBZ) and 98% (IVM+RBZ), while the efficacy against Haemonchus spp. was 0% (IVM), 97% (RBZ) and 100% (IVM+RBZ). The combination was the only treatment that achieved 100% clinical efficacy against IVM-resistant Haemonchus spp. The total egg excretion was reduced to 49.9% (IVM alone group), 6.3% (RBZ alone group) and 1.8% (IVM+RBZ combined group) compared to the untreated control. Although the combined treatment did not significantly increase the overall clinical efficacy in the current natural field conditions, an additive effect was achieved against IVM-resistant nematodes. In fact, the combination obtained significantly higher efficacy against IVM-resistant Haemonchus spp. than RBZ alone. Additionally, the epidemiological relevance of the reduction in the number of eggs excreted following the combined treatment is not negligible and should be taken into account in future studies. Further work is required to understand the advantages of nematodicidal combinations in different natural anthelmintic resistance scenarios.

Anthelmintic resistance in grazing beef cattle in central and northeastern areas of Argentina - An update.

The presence of anthelmintic resistance in Argentina has experienced a marked increase in cattle, with numerous reports showing levels of resistance of different parasite genera to different chemical groups. The aim of this study is to update comprehensively the situation of anthelmintic resistance to the different chemical groups in the most important areas of cattle production in Argentina. The study involved the determination of anthelmintic resistance in 62 cattle farms in 7 provinces using the faecal egg count reductions test. The results showed a marked increase of anthelmintic resistance compared to previous reports; the main resistant genera were Cooperia and Haemonchus to ivermectin, Ostertagia and Cooperia to ricobendazole, and Haemonchus to fenbendazole. There was also a distinct difference in clinical efficacies between subcutaneous ricobendazole and oral fenbendazole in favour of the latter, probably attributed to the administration route. Levamisole has showed high efficacy and broad antiparasitic spectrum. Anthelmintic resistance is widely and firmly established in grazing cattle production systems in the country; the diagnosis of resistance must be done in every particular farm in order to design a sustainable parasite control based on anthelmintics use.

The immature stages of Dermestes maculatus, Sarcophaga sp. and Phaenicia sericata as potential paratenic hosts for Trichinella spiralis in nature.

The role of some insect populations in the transmission of Trichinella sp. has been demonstrated. However, most of the studies have been conducted under controlled conditions which may influence the real role that they could play as a paratenic host in nature. To enlight this issue, a series of studies to determine the infective capability of the muscle larva of Trichinella spiralis recovered from immature stages of insect populations that fed on infected tissues exposed to natural conditions were carried out. Mice harbouring T. spiralis muscle larvae (ML) were sacrificed and deposited on a pitfall trap which was established in an open and safe area through 25 days in summer. Necrophilous and necrophagous insects that fed on corpses were recovered, identified and processed to search for live ML of T. spiralis. A complementary study in which maggots of Dermestes maculatus recovered from nature were induced to feed on muscle tissues harbouring T. spiralis larvae was also performed. The muscle larvae recovered from insects at different times of exposition were counted and inoculated to mice to determine the reproductive capability index (RCI). At day 3 of exposition, 29 live ML of T. spiralis were recovered from maggots of Phaenicia sericata. The RCI for these larvae was 133.6. On day 5 of exposition, maggots of Sarcophaga sp. were identified and 17 live T. spiralis larvae were recovered; the RCI of these larvae was 43.4. The T. spiralis ML recovered from maggots of D. maculatus obtained after 2 days of feeding on experimentally infected tissue showed a RCI of 24. The results suggest that larval stages of P. sericata, Sarcophaga sp. and D. maculatus might have an important role as a paratenic host of T. spiralis, which, in terms, may influence the epidemiology of this nematode in endemic areas of trichinellosis.

Persistence of Trichinella spiralis muscle larvae in natural decaying mice.

The influence of natural weather conditions on the viability and reproductive capability of Trichinella spiralis muscle larvae in mouse corpses exposed to summer and winter conditions in the Buenos Aires Province, Argentina, was studied. For this purpose, a total of 49 mouse corpses harbouring muscle larvae of T. spiralis were exposed for a period of 1, 2, 4 and 6 weeks in each of the seasons. Control corpses maintained at 8°C were also included. In summer, T. spiralis muscle larvae were recovered from corpses exposed up to 1 week only. The viability of these larvae was 54.2%, and the reproductive capability index in mice (RCI) was 13.1 and significantly lower than the control (p<0.0005). Morphologic deterioration and reduction in the glycogen content of cysts and larvae were observed at the second week of exposition. By week 4, larval stages of Dermestes maculatus were observed inside corpses, and 22 live muscle larvae of T. spiralis were obtained by artificial digestion of their bodies. In winter, T. spiralis muscle larvae were always recovered, the viability being almost 100% except for a significant reduction by week 6 of exposition (p<0.0001). For this season, the RCI were 50.5, 46.9, 59.7 and 45.2 for the periods of 1, 2, 4 and 6 weeks of exposition, respectively. The morphology of cysts and larvae did not show alterations, and no variations were observed as well in glycogen reserves during the 6-week period of exposition. RCI of non-exposed muscle larvae were always significantly higher that any of those recorded from muscle larvae that belonged to exposed corpses (p=0.0005). The present results demonstrate that muscle larvae of T. spiralis are able to survive in nature and keep infective for a 1-week period in summer and at least for 6 weeks in winter, becoming an important source of infection for scavengers. In summer, larvae stages of D. maculatus, and probably other insects, may play an important role in the survival and transmission of T. spiralis in the sylvatic cycle.

Comparative efficacy of trichlorphon and trichlorphon/ivermectin combination treatment against anthelmintic-resistant cattle nematodes in Argentina.

Twenty-four Holando Argentino male calves were treated orally with 50 mg/kg body weight trichlorphon (TCF); 0.2 mg/kg body weight subcutaneous ivermectin (IVM); a combination of TCF+IVM at the same doses and administration routes; or remained untreated (control group). All calves were necropsied at day 14 post treatment for counting and identification of worms from abomasum, small and large intestines and lungs to determine the absolute efficacy (controlled efficacy test) for each treatment. Using the faecal egg count reduction test, the efficacy was 63.7 % for TCF, 72.3 % for IVM and 99.2 % for TCF+IVM. The absolute efficacy of IVM and IVM+TCF was 100 % against Haemonchus placei, Trichostrongylus axei and Ostertagia ostertagi (p < 0.05). TCF showed a similar level of efficacy except against O. ostertagi (84.7 %). Efficacy of the treatments against Cooperia oncophora/pectinata/mcmasteri was 80.4 % for IVM, 95.7 % for TCF and 99.6 % for TCF+IVM; against Trichostrongylus colubriformis was 79 % for IVM, 86.2 % for TCF and 94.1 % for TCF+IVM; against Nematodirus helvetianus was 0 % for IVM, 100 % for TCF and 93.8 % for TCF+IVM. The efficacies of TCF, IVM and TCF+IVM were 100 % against Oesophagostomum radiatum and Trichuris spp. The efficacy of TCF against Dictyocaulus viviparus was 52 % and 100 % for IVM and IVM+TCF, respectively. This is the first report of Trichostrongylus colubriformis and Nematodirus helvetianus resistant to ivermectin treatment in cattle of Argentina. The TCF+IVM combination could be an alternative for the control and treatment of nematode infections including IVM-resistant strains.

The efficacy of trichlorphon and naphthalophos against multiple anthelmintic-resistant nematodes of naturally infected sheep in Argentina.

An anthelmintic efficacy trial was conducted in sheep harbouring anthelmintic-resistant worms in Argentina. Seventy lambs were selected from a flock that had been grazed on pastures infected with trichostrongyles previously shown to be resistant to the main anthelmintic groups. Lambs were allocated to comparable groups of ten animals each and treated with trichlorphon (50 mg/kg body weight (b.w.) orally); naphthalophos (50 mg/kg b.w. orally); ivermectin (0.2 mg/kg b.w. subcutaneously); fenbendazole (5 mg/kg b.w. orally); levamisole (8 mg/kg b.w. subcutaneously) and closantel (10 mg/kg b.w. orally). There was also an untreated group. The dose selection was based on manufacturer's recommendations.Faecal samples were collected 0 and 10 days post treatment to estimate efficacy (faecal egg count reduction). Six animals from each group were necropsied at day 10 for enumeration/identification of worms from the abomasum, small and large intestines to determine the absolute efficacy of each agent (controlled efficacy test). Trichlorphon and naphthalophos were effective (> 99 %) against Haemonchus contortus (p < 0.05).Naphthalophos also showed efficacy against Trichostrongylus axei (99.3 %), Teladorsagia circumcincta (97.8 %), Trichostrongylus colubriformis (99.2 %), Cooperia punctata/curticei/pectinata (90.4 %), Nematodirus spathiger (89.2 %) and Oesophagostomum venulosum/columbianum (93.7 %). Fenbendazole and levamisole showed efficacy (> 95 %) against all nematodes except T. colubriformis. The efficacy of ivermectin was low against H. contortus (23 %) and Cooperia spp. (46.3 %). Closantel showed low efficacy against T. axei (64.4 %), H. contortus (80.6 %) and T. colubriformis (59.5 %).When anthelmintic resistance is widespread, trichlorphon treatment is appropriate if H. contortus is present; however, naphthalophos represents an effective therapeutic alternative for incorporation into worm control programmes.

The efficacy of monepantel, an amino-acetonitrile derivative, against gastrointestinal nematodes of sheep in three countries of southern Latin America.

The efficacy of the novel anthelmintic, monepantel (an amino-acetonitrile derivative), was investigated in sheep naturally infected with gastrointestinal nematodes in five studies in Argentina, Brazil, and Uruguay. Monepantel, administered at 2.5 mg/kg liveweight, was highly effective (>99.7%) against Haemonchus contortus, Teladorsagia (Ostertagia) circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Cooperia curticei, Cooperia mcmasteri, Cooperia oncophora, Cooperia pectinata, Cooperia punctata, and Nematodirus spathiger, including strains resistant to the older broad-spectrum anthelmintics. Efficacy against C. mcmasteri, C. pectinata, and C. punctata is documented for the first time. The treatment with monepantel was well tolerated by the sheep.