A cysteine protease from the latex of Ficus benjamina has in vitro anthelmintic activity against Haemonchus contortus.

Haemonchus contortus is a gastrointestinal nematode that is responsible for high mortality rates in ruminant herds. The resistance of nematodes to synthetic anthelmintics is widespread and requires a continuous search for new bioactive molecules, such as proteins. The objective of this study was to evaluate the anthelmintic potential of a protease purified from the latex of Ficus benjamina against H. contortus . Fresh latex was collected from plants via small incisions in the green stems, the rubber was removed by centrifugation, and the latex protein extract (LPE) was obtained. After LPE fractionation with ammonium sulfate and chromatography of the fraction containing the highest proteolytic activity on CM-cellulose, a cysteine protease (FbP) was purified. FbP has a molecular mass of approximately 23.97 kDa, and its proteolytic activity was stable between pH 6.0 and pH 10 and over a broad temperature range, with optimum activity at 60 °C. FbP inhibited both the development and exsheathment of H. contortus larvae, with 50% effective concentrations of 0.26 and 0.79 mg/mL, respectively. We conclude that this cysteine protease from F. benjamina latex with anthelmintic activity against H. contortus could be a promising alternative for the development of products for use in parasite control programmes.

Establishment of an Artificial Tick Feeding System to Study Theileria lestoquardi Infection.

The establishment of good experimental models for Theileria sp. infection is important for theileriosis research. Routinely, infection of ticks is accomplished by feeding on parasite-infected animals (sheep, cows and horses), which raises practical and ethical problems, driving the search for alternative methods of tick infection. Artificial tick feeding systems are based mainly on rearing ticks on host-derived or hand-made artificial membranes. We developed a modified feeding assay for infecting nymphal stages of Hyalomma anatolicum ticks with Theileria lestoquardi, a highly pathogenic parasite of sheep. We compared two different membranes: an artificial silicone membrane and a natural alternative using mouse skin. We observed high attachment rates with mouse skin, whereas in vitro feeding of H. anatolicum nymphs on silicone membranes was unsuccessful. We could infect H. anatolicum nymphs with T. lestoquardi and the emerging adult ticks transmitted infective parasites to sheep. In contrast, similar infections with Rhipicephalus bursa, a representative tick with short mouth-parts that was proposed as a vector for T. lestoquardi, appeared not to be a competent vector tick species. This is the first report of an experimentally controlled infection of H. anatolicum with T. lestoquardi and opens avenues to explore tick-parasite dynamics in detail.

Chemical constituents of Calotropis procera latex and ultrastructural effects on Haemonchus contortus / Constituintes químicos do látex de Calotropis procera e efeitos ultraestruturais em Haemonchus contortus

Abstract This study aimed to evaluate the anthelmintic and ultrastructural effects of Calotropis procera latex on Haemonchus contortus. C. procera latex was twice centrifuged at 10,000×g and dialyzed to obtain a fraction rich in proteins, named LP (latex protein), and at 3,000 rpm to obtain a fraction rich in secondary metabolites, named LNP (latex non-protein). Specimens of H. contortus exposed to LNP, LP and PBS in the Adult Worm Motility Test (AWMT) were submitted to scanning (SEM) and transmission (TEM) electron microscopy to verify changes in their ultrastructure. Phytochemical tests in the LNP indicated the presence of phenols, steroids, alkaloids and cardenolides. High-Performance Liquid Chromatography (HPLC) characterized the presence of the compounds gallic acid and quercetin in the LNP. The protein content in the LP was 43.1 ± 1.1 mg/mL and 7.7 ± 0.3 mg/mL in LNP. In AWMT, LNP and LP inhibited the motility of 100% of the nematodes, with LNP being more effective than LP and ivermectin more effective than both (p <0.05). Cuticle changes were observed by SEM and TEM in nematodes treated with LP and LNP. Calotropis procera latex has anthelmintic effects against H. contortus, causing damage to its cuticle and other alterations in its ultrastructure.

Resumo Este estudo objetivou avaliar os efeitos anti-helmínticos e ultraestruturais do látex de Calotropis procera sobre Haemonchus contortus. Látex de C. procera foi centrifugado duas vezes à a 10.000xg e dialisado para obter uma fração rica em proteínas, denominada proteínas do látex (LP). E centrifugado e centrifugado a 3.000 rpm, para obter uma fração rica em metabólitos secundários, denominada LNP (látex não proteico). Espécimes de H. contortus expostos à LNP, LP e PBS no Teste de Motilidade dos Nematoides Adultos (TMNA) foram submetidos a microscopia eletrônica de varredura (MEV) e de transmissão (MET), para verificar alterações em sua ultraestrutura. Testes fitoquímicos em LNP indicaram a presença de fenóis, esteroides, alcaloides e cardenolídeos. A presença dos compostos ácido gálico e quercetina em LNP foi caracterizada por Cromatografia Líquida de Alta Eficiência (CLAE). O conteúdo de proteínas em LP foi de 43,1 ± 1,1 mg/mL e de 7,7 ± 0,3 mg/mL em LNP. No TMNA, LNP e LP inibiram a motilidade de 100% dos nematoides, sendo LNP mais eficaz que LP, e a ivermectina mais eficaz que ambos (p <0,05). Alterações na cutícula de nematoides tratados com LP e LNP foram observadas por MEV e MET. O látex de C. procera apresenta efeito anti-helmíntico sobre H. contortus, causando danos à sua cutícula e outras alterações em sua ultraestrutura.

Ruminal fermentation and degradation patterns, protozoa population and urinary purine derivatives excretion in goats and wethers fed diets based on olive leaves.

Olives leaves, accrued during the processing of olive harvests for oil extraction, are poor in N, rich in crude fat and ADF (1.19, 8.03 and 28.2 g/100 g of DM, respectively), and relatively low in condensed tannins (11.1 mg/g of DM). Three experiments were conducted in a 2 x 3 (two animal species: goats vs. wethers; and three experimental diets: olive leaves without or with polyethylene glycol supply and olive leaves supplemented with barley and faba beans) factorial design to evaluate ruminal degradation and passage kinetics (Exp. 1), fermentation pattern and protozoa population (Exp. 2), and urinary purine derivatives excretion (Exp. 3). Polyethylene glycol was supplied to evaluate the effects of condensed tannins contained in olive leaves. Ruminal degradability of CP was low in both goats and wethers, although goats showed higher (P < 0.05) values than wethers. Supplementation of olive leaves with barley and faba beans increased (P < 0.001) ruminal degradability of DM and CP. Both goats and wethers fed olive leaves showed similarly low particulate fractional passage rates (0.021 and 0.023/h, respectively). Ingestion of olive leaves promoted low NH3-N and VFA concentrations, which reflect poor microbial activity. These concentrations, especially that of VFA, increased when barley and faba beans were added. Ingestion of olive leaves affected ruminal protozoa: Entodiniomorphida showed low concentrations and Holotricha completely disappeared. When animals received a diet based on olive leaves, barley, and faba beans, Holotricha appeared in the ruminal liquor and Entodiniomorphida increased (P < 0.001). In goats and wethers fed olive leaves alone, urinary allantoin excretion was very low (163 and 164 micromol/kg BW0.75 in goats and wethers, respectively), and moderate values (352 and 389 micromol/kg BW0.75 in goats and wethers, respectively) were observed when a diet of olive leaves, barley, and faba beans was fed. The polyethylene glycol supply did not have an effect in goats or in wethers, indicating the lack of an effect of condensed tannins in olive leaves. Ingestion of olive leaves promotes a low microbial activity, although its supplementation with readily degraded carbohydrates and protein improves microbial activity and, as a consequence, increases its ruminal degradation. In general, for most of the measured variables, there were no animal species x diet interactions. Thus, goats and wethers had similar ruminal activities when fed diets based on olive leaves.

A cysteine protease from the latex of Ficus benjamina has in vitro anthelmintic activity against Haemonchus contortus / Atividade anti-helmíntica in vitro de uma protease cisteínica do látex de Ficus benjamina contra Haemonchus contortus

Abstract Haemonchus contortus is a gastrointestinal nematode that is responsible for high mortality rates in ruminant herds. The resistance of nematodes to synthetic anthelmintics is widespread and requires a continuous search for new bioactive molecules, such as proteins. The objective of this study was to evaluate the anthelmintic potential of a protease purified from the latex of Ficus benjamina against H. contortus . Fresh latex was collected from plants via small incisions in the green stems, the rubber was removed by centrifugation, and the latex protein extract (LPE) was obtained. After LPE fractionation with ammonium sulfate and chromatography of the fraction containing the highest proteolytic activity on CM-cellulose, a cysteine protease (FbP) was purified. FbP has a molecular mass of approximately 23.97 kDa, and its proteolytic activity was stable between pH 6.0 and pH 10 and over a broad temperature range, with optimum activity at 60 °C. FbP inhibited both the development and exsheathment of H. contortus larvae, with 50% effective concentrations of 0.26 and 0.79 mg/mL, respectively. We conclude that this cysteine protease from F. benjamina latex with anthelmintic activity against H. contortus could be a promising alternative for the development of products for use in parasite control programmes.

Resumo Haemonchus contortus é um nematoide gastrintestinal, responsável por altas taxas de mortalidade em rebanhos de pequenos ruminantes. A resistência dos nematoides aos anti-helmínticos sintéticos está generalizada e requer uma busca contínua por novos compostos bioativos, como as proteínas. O objetivo deste trabalho foi avaliar o potencial anti-helmíntico da protease purificada do látex de Ficus benjamina contra H. contortus . O látex fresco foi coletado das plantas por pequenas incisões nas hastes verdes e o extrato proteico de látex (EPL) foi obtido. Após o fracionamento do EPL com sulfato de amônio e cromatografia da fração contendo a maior atividade proteolítica da CM-Celulose, uma protease cisteínica (FbP) foi purificada. A FbP tem massa molecular de cerca de 23,97 kDa, a atividade proteolítica foi estável entre pH 6,0 e pH 10 e ao longo de uma ampla faixa de temperatura, com atividade ótima a 60 °C. A FbP inibiu tanto o desenvolvimento quanto o desembainhamento das larvas de H. contortus, com 50% de inibição nas concentrações de 0,26 e 0,79 mg/mL, respectivamente. Concluímos que esta protease cisteínica do látex de F. benjamina, com ação anti-helmíntica contra H. contortus, pode ser uma alternativa promissora para o desenvolvimento de produtos a serem utilizados em programas de controle de parasitos.

Regeneration of cryoresistance of in vitro rumen ciliate cultures.

The purpose of this study was to investigate factors affecting mechanical- and cryo-resistance of the rumen ciliates Entodinium caudatum (E.c.), Entodinium furca monolobum (E.f.m.), Entodinium simplex (E.s.), Diplodinium denticulatum (two clones, D.d.01 and D.d.02), Diploplastron affine (D.a.) and Epidinium ecaudatum forma caudatum (E.e.c.) after long-term in vitro cultivation. Following prolonged in vitro cultivation (more than six months), the ciliates were very sensitive to both centrifugation and 5% (v/v) dimethylsulphoxide, with motility decreased to: 39 and 23% for E.c., 66 and 32% for E.f.m., 46 and 27% for D.d. 01, 64 and 41% for D.a., and 44 and 28% for E.e.c., respectively. Thus, cryopreservation was unsuccessful. The effect of supplementing the ciliate growth medium with rumen fluid, glycine-betaine, proline, myo-inositol, linoleic acid, Sel-Plex or insulin, together with the effect of the source of rumen fluid on ciliate resistance to centrifugation, dimethylsulphoxide and freezing was also tested. The omission of rumen fluid from the growth medium resulted in the loss of cryoresistance after one-month cultivation. Supplementing the growth environment with a combination of glycine-betaine, proline, linoleic acid, Sel-Plex, insulin plus improved quality rumen fluid significantly enhanced survival of the ciliates after the freezing-thawing procedure (from 1 to 33% survival in un-supplemented vs. supplemented for E.c., P<0.01; 4-40% E.f.m., P<0.01; 0-17% D.d., P<0.05; 5-7% D.a. and 4-36% E.e.c., P<0.01).

Stereocan observations on the surface topography of Gastrothylax crumenifer (Creplin, 1847) Poirier, 1883 and Paramphistomum epiclitum Fischoeder, 1904 (Trematoda: Digenea).

The critical-point-dried specimens of Gastrothylax crumenifer (Creplin, 1847) Poirier, 1883 and Paramphistomum epiclitum Fischoeder, 1904, both recovered from the rumen of sheep, were studied by scanning electron microscopy at magnifications ranging from 10 to 10,000x to reveal the structural differences of the tegument between the two species. In G. crumenifer, both dorsal and ventral surfaces have tubercle-like tegumental elevations, devoid of spines. A row of prominent, regularly arranged, button-like protuberances encircle the rim of mouth. It is suggested that these are sensory in nature. The lining of the buccal tube also bears papillae, some of which appear balloon-like. The tegument near the outer acetabular rim is thrown into prominent ridges bearing groups of six to eight small papilla-like elevations. Towards the interior of the acetabular cavity this pattern merges into one with stout, finger-like projections with groups of papillae at their blunt tips. From their structure, these projections appear to aid strong anchorage to the host's surface and the papillae to be secretory. P. epiclitum has a similar tuberculated pattern on the dorsal and ventral surfaces. In the oral region, the tegument has some irregular, patch-like elevated areas. The acetabular surface is thrown into a pattern of deep folds and craters.

The rate of uptake and metabolism of starch grains and cellulose particles by Entodinium species, Eudiplodinium maggii, some other entodiniomorphid protozoa and natural protozoal populations taken from the ovine rumen.

The rates of engulfment and breakdown of starch grains and cellulose particles and of the rate of synthesis of amylopectin from cellulose by individual species of entodiniomorphid protozoa (grown in vivo and in vitro) and incubated anaerobically in vitro were studied. Rates of starch uptake varied from 2.3 to 770 micrograms/mg protozoal protein/min; the lowest was found with Diploplastron affine and the highest with Entodinium spp. on initial incubation with starch grains. The rate of starch breakdown varied from 0.49 to 8.6 micrograms/mg protein/min; the rate was dependent on the initial starch concentration inside the protozoa. Eudiplodinium maggii engulfed cellulose particles more rapidly (2-7 times) than rice starch grains and digested the cellulose at rates of 10 to 16.5 micrograms/mg protein/min. In a mixture of starch grains and cellulose particles, it engulfed the latter at 1.35 to 25 times the rate of the former. Eudiplodinium maggii and Epidinium caudatum, but not Entodinium spp. or Dip. affine, synthesized an amylopectin-like material from cellulose at rates of 0.4 to 4.75 micrograms/mg protein/min. If these reactions occur in the rumen in vivo, up to 9 g of amylopectin could be synthesized from cellulose each day by the entodiniomorphid protozoa.

Effect of ruminal inoculation of Isotricha alone or a mixed B-type fauna in a defaunated rumen on the digestion of a hay-maize diet (70:30) in sheep.

Two adult sheep (75 kg live weight) fitted with rumen cannulas were defaunated by the emptying method during the first period of the experiment. They were inoculated with the genus Isotricha alone during the second period, and with a mixed ciliate population (Entodinium, Eudiplodinium, Epidinium) during the third. They were fed a diet of grass hay (840 g) and pelleted maize grains (360 g) in 8 meals per day, every 3 h. Defaunation was successful and no accidental contamination occurred during the experiment. The protozoa had no significant effect on the volume of rumen digesta, nor on the turnover of the particulate phase. The addition of Isotricha and of the mixed fauna increased the ADF digestibility of the diet but, in the same animals, lowered the in sacco degradation of wheat straw. The ruminal pool sizes of dry matter (DM), organic matter (OM), nitrogen (N), neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid lignin detergent (ADL) remained unchanged after protozoa inoculations. The concentration of total volatile fatty acids (VFA) was not altered by faunation with Isotricha or a mixed fauna. The molar proportion of acetate increased at the expense of all the other VFAs (mainly propionate with the mixed fauna). Correspondingly, the proportion of methane in the rumen gases increased and that of CO2 decreased in inoculated animals. The ammonia concentration was highest in animals with a mixed fauna and lowest in those inoculated with Isotricha alone. This trend is explained in terms of the specific effect of the different genera of protozoa on nitrogen metabolism.

In vitro excystation of Cryptosporidium parvum.

Protocols for in vitro excystation of oocysts of Cryptosporidium parvum, including different chemical pre-incubation steps, were compared to examine some of the biochemical triggers involved in excystation and to define an in vitro excystation protocol of a reproducibly high efficiency. Pre-incubation steps which increased the permeability of the oocysts were found to enhance excystation dynamics and pre-treatment of oocysts with saliva was found to decrease the permeability and reduce excystation. Although excystation was maximal after incubation for 4 h, sporozoites tended to lyse over this period, and maximum sporozoite recovery occurred after 30 min. The results obtained are discussed in relation to excystation protocols adopted by different research groups and a number of recommendations are given for in vitro excystation of C. parvum oocysts.

Evidence and mechanisms of immunosuppression in tick infestations.

The abundance and ubiquity of ticks from ancient times long ago suggested that they have eluded host immunity. In the last 15 years, several authors have demonstrated suppression of the Th1 responses (cell-mediated immunity), and sometimes the Th2 responses (humoral immunity), subsequent to tick infestations in laboratory and natural models. Although the mechanisms to produce suppression are not well-defined yet, evidences for antigenic competition, lymphocyte cytotoxicity, presence of immuno-inhibiting substances in the saliva, and existence of modulators of cytokines in salivary extracts have been reported. Management of tick-induced immunosuppression is essential to replace tick control by acaricide application with more environmentally sound vaccination.

Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen.

AIMS: To determine the effect of condensed tannins in Calliandra calothyrsus (calliandra) on rumen microbial function. METHODS AND RESULTS: Microbial populations, ruminal protein synthesis and fermentation end-products were measured in sheep fed roughage hay supplemented with calliandra (30%), with and without inclusions of polyethylene glycol (PEG) to counteract the effect of tannin. Molecular and conventional enumeration techniques were used to quantify rumen bacteria, fungi and protozoa, and protein synthesis was predicted from estimates of urinary purine excretion. The total number of cellulolytic bacteria, including populations of Fibrobacter succinogenes and Ruminococcus spp., was significantly lower in sheep supplemented with calliandra and these populations increased when animals were treated with PEG. By contrast, protozoa and fungi and the microbial group containing Bacteroides-Porphyromonas-Prevotella bacteria appeared to be less affected. The efficiency of microbial protein synthesis in the rumen was not altered significantly. CONCLUSION: Calliandra caused significant shifts in rumen microbial populations without changing the efficiency of protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of calliandra tannins on rumen digestion may result more from complexing with nutrients than direct inhibition of micro-organisms.

Evaluación de la técnica inmunoensayo en capa delgada utilizando microplacas, en la detección de anticuerpos cirulantes de Oestrus ovis, en ovinos infestados naturalmente / Evaluation of the immunoassay test in thin layer, using microplates of polyestirene for the detection of circulating antibodies of Oestrus ovis naturally infected ovines

Introducción. La estrosis es una miasis cavitaria que afecta a los ovinos y caprinos. En la actualidad el diagnóstico es a través de la observación de los signos clínicos y el hallazgo de larvas en los conductos nasales de los animales a la necropsia. Diferentes técnicas serológicas se han utilizado y evaluado para tratar de realizar un mejor diagnóstico de esta enfermedad. Objetivo. Evaluar la prueba de inmunoensayo en capa delgada (ICD) utilizando microplacas de poliestireno, para el diagnóstico de estrosis ovina. Material y métodos. Se utilizaron 95 ovinos sacrificados para abasto, de los cuales se obtuvo el suero y se les realizó el examen postmorten para la detección de la presencia de larvas en los conductos nasales. La presencia de larvas en los conductos nasales fue considerada como prueba de oro. El antígeno usado fue a partir de larvas L2 con una concentración de proteína de 4.2 mg/mL. La prueba se realizó en microplacas de poliestireno de fondo plano y de 96 pozos, se siguió el mismo procedimiento utilizado en la técnica descrita por Bautista 1982. Se determinó la sensibilidad y se midió la concordancia entre ambas pruebas, calculando el valor de kappa. Resultados. De los 95 ovinos sacrificados, 51 (54 por ceinto) fueron positivos, a la preencia de larvas en el examen postmortem y 61 (51 por ciento) a la prueba de ICD y u valor de kappa de 0.53 al comparar ambas pruebas. Conclusiones. El uso de microplacas en la prueba ICD es una alternativa para el diagnóstico de estrosis ovina en estudio epidemiológicos ya que permite con una sensibilidad alta, analizar una mayor cantidad de sueros por placa, utilizando una menor cantidad de antígeno