Anthelmintic resistance: is a solution possible?

More than 50 years after anthelmintic resistance was first identified, its prevalence and impact on the animal production industry continues to increase across the world. The term "anthelmintic resistance" (AR) can be briefly defined as the reduction in efficacy of a certain dose of anthelmintic drugs (AH) in eliminating the presence of a parasite population that was previously susceptible. The main aim of this study is to examine anthelmintic resistance in domestic herbivores. There are numerous factors playing a role in the development of AR, but the most important is livestock management. The price of AH and the need to treat a high number of animals mean that farmers face significant costs in this regard, yet, since 1981, little progress has been made in the discovery of new molecules and the time and cost required to bring a new AH to market has increased dramatically in recent decades. Furthermore, resistance has also emerged for new AH, such as monepantel or derquantel. Consequently, ruminant parasitism cannot be controlled solely by using synthetic chemicals. A change in approach is needed, using a range of preventive measures in order to achieve a sustainable control programme. The use of nematophagous fungi or of plant extracts rich in compounds with anthelmintic properties, such as terpenes, condensed tannins, or flavonoids, represent potential alternatives. Nevertheless, although new approaches are showing promising results, there is still much to do. More research focused on the control of AR is needed.

Fatal Peritoneal Migration of Strongylus edentatus in a Foal.

A 7-month-old female mixed breed foal with a 2-day history of recumbency and inability to open its mouth convulsed acutely and died and was submitted for necropsy examination. The foal was thin and large patches of haemorrhage were present throughout the peritoneal wall, the diaphragmatic surfaces and the retroperitoneum. Numerous nematode larvae were visible on the serosal surfaces and penetrated and embedded into the subserosa associated with the haemorrhages. The dorsal portion of the abdominal diaphragm had a partial tear and large numbers of nematodes were within the muscle fibres. Histologically, the larvae had a smooth cuticle, polymyarian/coelomyarian musculature and multinucleated intestinal cells, and were typically surrounded by haemorrhage, neutrophils, dense fibrovascular connective tissue and rare multinucleated giant cells. Parasitological examination identified the larvae as Strongylus edentatus based on the morphology of the buccal capsule. Additionally, there was severe muscle necrosis of the tongue and liver tissue analysis detected selenium deficiency. S. edentatus infections are uncommon in California, USA, and are typically non-lethal. In this case, the selenium deficiency may have led to immunosuppression, resulting in the hyperinfection with S. edentatus, and to the muscle damage and tear of the diaphragm. Although ivermectin treatment was indicated in the history, inadequate deworming or anthelmintic resistance may have played a role in the severity of infection.

A cream containing omega-3-fatty acids, humectants and emollients as an aid in the treatment of equine Culicoides hypersensitivity.

BACKGROUND: Topical application of polyunsaturated fatty acids (PUFAs) has shown satisfactory results in dogs and humans with allergic skin diseases. Urea and glycolic acid act as keratolytics and moisturizers. Culicoides hypersensitivity is the most common equine hypersensitivity disorder and only limited treatment options exist. OBJECTIVES: To evaluate the effect of a cream containing topical PUFAs, humectants and emollients on clinical signs of equine Culicoides hypersensitivity. ANIMALS: Privately owned horses (n = 28) with clinical signs of Culicoides hypersensitivity. METHODS AND MATERIALS: For a period of four weeks, one half of the horse's body (left or right) was treated with a cream containing concentrated fish oil and several moisturizing and emollient ingredients in a randomized, single-blinded fashion to evaluate the influence of the treatment on skin lesions. In the subsequent four weeks, the lesional areas of the entire body were treated to assess the treatment effect on pruritus. Additionally, the quality of the hair coat, an overall assessment and adverse effects were recorded. RESULTS: Twenty-one horses completed the study. Skin lesions on the treated side improved significantly between days 0 and 28 (P < 0.0001) in comparison to the untreated side. Neither pruritus scores nor coat quality improved significantly between days 0 and 56. Overall condition improved during the study. Five horses showed adverse effects. CONCLUSIONS AND CLINICAL IMPORTANCE: The cream improved Culicoides-induced skin lesions in affected horses, but anti-pruritic effects were less prominent.

The establishment of in vitro culture and drug screening systems for a newly isolated strain of Trypanosoma equiperdum.

Dourine is caused by Trypanosoma equiperdum via coitus with an infected horse. Although dourine is distributed in Equidae worldwide and is listed as an internationally important animal disease by the World Organization for Animal Health (OIE), no effective treatment strategies have been established. In addition, there are no reports on drug discovery, because no drug screening system exists for this parasite. A new T. equiperdum strain was recently isolated from the genital organ of a stallion that showed typical symptoms of dourine. In the present study, we adapted T. equiperdum IVM-t1 from soft agarose media to HMI-9 liquid media to develop a drug screening assay for T. equiperdum. An intracellular ATP-based luciferase assay using CellTiter-Glo reagent and an intracellular dehydrogenase activity-based colorimetric assay using WTS-8 tetrazolium salt (CCK-8 reagent) were used in order to examine the trypanocidal effects of each compound. In addition, the IC50 values of 4 reference trypanocidal compounds (pentamidine, diminazene, suramin and melarsomine) were evaluated and compared using established assays. The IC50 values of these reference compounds corresponded well to previous studies involving other strains of T. equiperdum. The luciferase assay would be suitable for the mass screening of chemical libraries against T. equiperdum because it allows for the simple and rapid-evaluation of the trypanocidal activities of test compounds, while a simple, inexpensive colorimetric assay will be applicable in developing countries for the evaluation of the drug sensitivity of epidemic trypanosome strains.

SCANNING ELECTRON MICROSCOPIC CHANGES IN HABRONEMA MUSCAE AFTER IN VITRO EXPOSURE TO PLANT EXTRACT (VERBESINA ALTERNIFOLIA).

Scanning electron microscopy (SEM) was used to study the morphological alteration occurring in Habronema muscae adult female worms after in vitro exposure to different doses of Verbesina alternifolia oil extract. The half maximal lethal concentration (LC50) was reached 400 ppm after 24hrs, while LC100 was reached 600 ppm after 48hrs. Irreversible degenerative changes were recorded such as shrinking, detachment and distortion of the cuticle, cephalic and distal region. The cuticular surface had a wrinkled, corrugated appearance with longitudinal ridges and transverse thick folds. The lips and papillae were deformed and aggregated over each other. There is a direct relation between the level of the recorded degenerative changes and the increase in the dose and exposure time. In the same time no degen'erative changes were recorded in the control worm exposed to PBS till the end of the exposure period.

Equine antibody response to larval Parascaris equorum excretory-secretory products.

Parascaris equorum is an intestinal nematode of foals and young horses that can produce mild to severe pathology. Current diagnosis is limited to detection of patent infections, when parasite eggs are identified during fecal examinations. This study examined the use of larval P. equorum excretory-secretory (ES) products in a western blot test for diagnosis of prepatent equine P. equorum infection. Sera from adult mares negative for patent P. equorum infections, foals prior to consuming colostrum, and P. equorum infected foals were used as controls in this study. Study samples included sera from 18 broodmares prior to parturition and sera from their foals throughout the process of natural infection. Sera from study horses were examined for IgG(T) antibody recognition of ES products. Foals naturally infected with P. equorum possessed IgG(T) antibodies against 19kDa, 22kDa, 26kDa, and 34kDa ES products. However, passive transfer of colostral antibodies from mares was shown to preclude the use of the crude larval ES product-based western blot test for diagnosis of prepatent P. equorum infections in foals.

Serological investigation of transplacental infection with Neospora hughesi and Sarcocystis neurona in broodmares.

The aim of the present study was to investigate the likelihood of transplacental transmission of Neospora hughesi and Sarcocystis neurona in foals, born from seropositive mares. Three broodmares with persistent N. hughesi infection gave birth to eight healthy foals over a period of 7 years. These foals were seropositive to N. hughesi prior to colostrum ingestion, with titers ranging between 640 and 20,480, measured by indirect fluorescence antibody test (IFAT). Of 174 foals born at another farm to mares with a high seroprevalence to S. neurona, only one (with a pre-colostrum antibody titer of 80) tested seropositive. Transplacental transmission of N. hughesi seems to occur from latently infected mares to their foals, while this route of transmission does not seem to occur commonly for S. neurona.

How does the suppression of energy supplementation affect herbage intake, performance and parasitism in lactating saddle mares?

Agroecology opens up new perspectives for the design of sustainable farming systems by using the stimulation of natural processes to reduce the inputs needed for production. In horse farming systems, the challenge is to maximize the proportion of forages in the diet, and to develop alternatives to synthetic chemical drugs for controlling gastrointestinal nematodes. Lactating saddle mares, with high nutritional requirements, are commonly supplemented with concentrates at pasture, although the influence of energy supplementation on voluntary intake, performance and immune response against parasites has not yet been quantified. In a 4-month study, 16 lactating mares experimentally infected with cyathostome larvae either received a daily supplement of barley (60% of energy requirements for lactation) or were non-supplemented. The mares were rotationally grazed on permanent pastures over three vegetation cycles. All the mares met their energy requirements and maintained their body condition score higher than 3. In both treatments, they produced foals with a satisfying growth rate (cycle 1: 1293 g/day; cycle 2: 1029 g/day; cycle 3: 559 g/day) and conformation (according to measurements of height at withers and cannon bone width at 11 months). Parasite egg excretion by mares increased in both groups during the grazing season (from 150 to 2011 epg), independently of whether they were supplemented or not. This suggests that energy supplementation did not improve mare ability to regulate parasite burden. Under unlimited herbage conditions, grass dry matter intake by supplemented mares remained stable around 22.6 g DM/kg LW per day (i.e. 13.5 kg DM/al per day), whereas non-supplemented mares increased voluntary intake from 22.6 to 28.0 g DM/kg LW per day (13.5 to 17.2 kg DM/al per day) between mid-June and the end of August. Hence total digestible dry matter intake and net energy intake did not significantly differ between supplemented and non-supplemented mares during the second and third cycles. In conclusion, supplementing lactating mares at pasture should not be systematic because their adaptive capacities enable to increase herbage intake and ensure foal growth. Further research is needed to determine the herbage allowance threshold below which supplementation is required.

L-cysteine replaces microaerophilous culture conditions for the in vitro initiation of Theileria equi.

Fifty-one blood samples of carrier horses from Theileria equi-endemic localities in South Africa were used for two different methods of in vitro culture initiation of T. equi parasites. Cultures were initiated either in a oxygen-reduced gas mixture or in a 5% CO2-in-air atmosphere in combination with L-cysteine-supplemented culture medium. Out of the 51 blood samples, 43 and 42 cultures, respectively, became culture positive. A possible explanation for this observation is proposed.

Observations on effects of a neem seed extract (MiteStop®) on biting lice (mallophages) and bloodsucking insects parasitizing horses.

The hair of 300 horses belonging to short hair and long hair races had been routinely treated during the last 3 years with a neem seed extract (MiteStop®) in order to kill mallophages (e.g., specimens of the genus Werneckiella). It was found that in all cases, a hidden infestation with these biting lice had existed, which became visible when the product (diluted 1:20 with tap water) was brushed onto the hair. The mallophages left the body surface and became visible as a fine "wooly looking" layer at the tips of the hair. Furthermore, this treatment stopped the forming of dandruff of the skin of the horses, which, in case of heavy mallophage infestations, had looked like being powdered. Another interesting result of the treatment was reported by the riders. They described that the product had a considerable repellent effect on bloodsucking tabanids, mosquitoes, ceratopogonids, simuliids, as well as on licking flies. This repellency effect was noted to last for up to 7 days if the horses were not washed.

Endogenous transplacental transmission of Neospora hughesi in naturally infected horses.

Over a 2-yr study period, we investigated possible endogenous transplacental transmission of Neospora hughesi in 74 mare and foal pairs following the diagnosis of neuronal neosporosis in a weanling foal. Presuckle and postsuckle serum of each foal, serum and colostrum of each periparturient mare, and serum of each mare and foal pair, collected at 3-mo intervals thereafter, were tested for N. hughesi using an indirect fluorescent antibody test (IFAT). Furthermore, whole blood and colostrum samples and placentae were tested for the presence of N. hughesi by real-time PCR. The mares' seroprevalence at foaling based on IFAT (titer ≥ 160) was 52 and 6% in 2006 and 2007, respectively. Colostral antibodies against N. hughesi were detected in 96 and 11% of the mares in the 2-yr study. With the exception of 3 foals, all remaining foals were born seronegative to N. hughesi. Passive transfer of colostral antibodies to N. hughesi was documented in 15 foals. Three foals born from 2 different mares had presuckle antibodies at a titer ranging from 2,560 to 20,480. All 3 foals were born healthy. Two foals were born to the same dam that also gave birth to the weanling diagnosed with neuronal neosporosis in 2005. The third foal was born to a second mare with no previous foaling history at the farm. Seroconversion was documented in 10 foals and 9 mares over the 2-yr study. All blood and colostrum samples tested PCR negative for N. hughesi. Only 1 placenta collected in 2007 from the mare with the 2 congenitally infected foals tested PCR positive for N. hughesi. In conclusion, N. hughesi persisted in this population via endogenous transplacental infection.

Repellent activity of plant-derived compounds against Amblyomma cajennense (Acari: Ixodidae) nymphs.

Repellence responses of Amblyomma cajennense nymphs to callicarpenal, intermedeol, Hyptis suaveolens essential oil, extract of Melia azedarach, Cymbopogon nardus, Spiranthera odoratissima, Chenopodium ambrosioides, Ageratum conyzoides, Mentha pulegium, Ruta graveolens, and Memora nodosa were studied. Among these the extract of C. nardus stood out because of the long-lasting repellence, maintaining, in the highest concentration, 35h of protection against 90% of the nymphs. The essential oil of H. suaveolens and the extracts of C. ambrosioides and A. conyzoides showed good repellence index (66%) when applied in high concentrations. However, greater protection could be obtained at higher concentrations but with a shorter repellence time. Callicarpenal, intermedeol, extract of M. Pulegium, and M. nodosa leaves showed moderate repellence in high concentrations. Extracts from M. azedarach, R. graveolens, S. odoratissima, and M. nodosa roots showed little or no repellent effect. These results show that some plant extracts may represent a promising alternative in the control of infestations by A. cajennense.

Parascaris equorum in foals and in their environment on a Swedish stud farm, with notes on treatment failure of ivermectin.

Environmental contamination and the egg excretion pattern of the ascarid Parascaris equorum (Nematoda) was investigated in relation to anthelmintic treatment on a Swedish stud farm. Faecal samples from 15 foals, dewormed every 8th-week with a paste formulation of ivermectin at the standard dose rate of 0.2 mg/kg bodyweight, were collected at five sampling occasions between August and November 2006. In addition, soil samples were obtained from four paddocks used by these foals in November 2006. The number of eggs per gram (epg) was counted in both faeces and soil. Egg excretion started when the foals were 3-4 months, and reached the highest levels when they were approximately 5-month-old, and was then followed by a decline. Egg excretion seemed to be unaffected by ivermectin despite these foals were dewormed at regular intervals. In four out of five foals examined 10 days after treatment, epg actually increased. In contrast, when either fenbendazol or pyrantel embonate were used instead of ivermectin, treatments were effective. The number of eggs in soil was significantly higher in the permanent paddock compared to in the temporarily used soil paddock and in the summer paddocks.

Molecular evidence for transplacental transmission of Theileria equi from carrier mares to their apparently healthy foals.

The intra-erythrocytic parasite Theileria equi is one of two tick-transmitted causative agents of equine piroplasmosis. Piroplasms of T. equi can be transmitted across the equine placenta and once a horse is infected, it appears to remain a lifelong carrier, since anti-theilerial drugs suppress but do not eliminate the parasite. Carrier mares may transmit the organism to their offspring and this may result in abortion or neonatal piroplasmosis, but observations by some researchers suggest that foals may be born as carriers yet remain apparently healthy. Using a T. equi-specific oligonucleotide probe, we have determined that transplacental transmission occurs early in equine foetal development and that carrier mares may give birth to healthy carrier foals. Investigation of parasite levels and the effect of maternal colostrum on the newborn suggests that colostral T. equi antibody may act to suppress parasitaemia in the newborn, reducing the incidence of clinical neonatal piroplasmosis.

Equine Cryptosporidium parvum infections in western Poland.

A total of 564 fecal specimens from 318 horses used for recreational riding, child hippotherapy, and racing at ten commercial and government-run stables in western Poland were tested for Cryptosporidium parvum oocysts by microscopic examination of Ziehl-Neelsen stained smears, enzyme immunoassay, and combined direct immunofluorescent antibody and fluorescent in situ hybridization. Also, seven stool specimens from five personnel who had repeated contact with these horses were tested for C. parvum oocysts. Eleven horses that shed C. parvum oocysts were found in five of ten stables (50%). The prevalence of infection varied from 0% to 11.5%. The overall prevalence of equine C. parvum-associated cryptosporidiosis in the Wielkopolska region of western Poland was 3.5%. C. parvum oocysts were found only in fecal samples from mature horses, the number of oocysts was low, and infections were not associated with clinical signs. Oocysts were not found in human fecal specimens.

Risk of transplacental transmission of Sarcocystis neurona and Neospora hughesi in California horses.

The study objective was to assess the risk of transplacental transmission of Sarcocystis neurona and Neospora hughesi in foals from 4 California farms during 3 foaling seasons. Serum of presuckle foals and serum and colostrum of periparturient mares were tested using indirect fluorescent antibody tests for S. neurona and N. hughesi. Serum antibody titers were < or =10 in 366 presuckle foals tested. There was no serologic or histologic evidence of either parasite in aborted fetuses or placentas examined. Positivity for S. neurona and N. hughesi in mares increased with age. Mares < or =9 yr that originated from Kentucky were 3.8 and 1.4 times more likely to be positive for S. neurona and N. hughesi, respectively, than mares from California. The strength of association between positivity to either parasite and state of birth decreased as age increased. Mares positive for S. neurona and N. hughesi were 2.2 and 1.7 times more likely, respectively, to have a previous abortion than negative mares, adjusted for age and state of birth. The annual mortality rate for mares was 4%. The annual incidence rate of equine protozoal myeloencephalitis was 0.2%. In conclusion, there was no detectable risk of transplacental transmission of S. neurona and N. hughesi. Prevalence of antibodies against both parasites in mares increased with age.

Interpretation of the detection of Sarcocystis neurona antibodies in the serum of young horses.

Horses that are exposed to Sarcocystis neurona, a causative agent of equine protozoal myeloencephalitis, produce antibodies that are detectable in serum by western blot (WB). A positive test is indicative of exposure to the organism. Positive tests in young horses can be complicated by the presence of maternal antibodies. Passive transfer of maternal antibodies to S. neurona from seropositive mares to their foals was evaluated. Foals were sampled at birth (presuckle), at 24h of age (postsuckle), and at monthly intervals. All foals sampled before suckling were seronegative. Thirty-three foals from 33 seropositive mares became seropositive with colostrum ingestion at 24h of age, confirming that passive transfer of S. neurona maternal antibodies occurs. Thirty-one of the 33 foals became seronegative by 9 months of age, with a mean seronegative conversion time of 4.2 months. These results indicate that evaluation of exposure to S. neurona by WB analysis of serum may be misleading in young horses.

Ocular Halicephalobus (syn. Micronema) deletrix in a horse.

Ocular contents from a horse with a 4-week history of severe unilateral uveitis were submitted for histopathologic examination. A severe unilateral granulomatous chorioretinitis with intralesional Halicephalobus deletrix was diagnosed. The horse developed progressive neurologic signs several days following the surgery to remove ocular contents and implant a prosthesis and was subsequently euthanatized. A severe multifocal granulomatous encephalitis with intralesional H. deletrix, localized primarily to the optic chiasm, thalamus, and brain stem, was diagnosed from tissues acquired at necropsy. The other eye was not affected. This is the first report of ocular parasitism by H. deletrix and suggests possible systemic dissemination from a primary site in the eye.