The anthelmintic effects of the ethanol extract of Terminalia catappa L. leaves against the ruminant gut parasite, Fischoederius cobboldi.

Presently, no effective anthelmintic drugs have been used to treat and control paramphistomosis, a severe disease of ruminants. In this study, we have investigated the in vitro anthelmintic effect of the leaves of Terminalia catappa L. crude extract (TcCE) and albendazole (ABZ) on adult Fischoederius cobboldi after incubating the flukes in RPMI-1640 medium containing the TcCE at various doses and times. The TcCE-treated flukes at all dosages exhibited rapid decrease of motility, and the relative motility (RM) values were decreased sharply from start to 3 h. Worms were killed after 6 and 12 h of treatment with 1000, 1500 and 2000 µg mL(-1) as well as 500 µg mL(-1) of TcCE, respectively. By light microscopy examination, the flukes exhibited the earliest alteration in a limited area of the tegument. At scanning electron microscopy level, the flukes' tegument showed similar sequence of morphological alterations after treatment with ABZ and TcCE that consisted of swelling of ridges and folds, followed by blebbing and rupturing of the blebs, leading to the erosion, lesion and disruption of the tegument. Hence, in vivo studies should be performed to examine whether the TcCE may serve as a powerful anthelmintic drug for treatment of paramphistomosis.

Chemo-profiling and bioassay of phytoextracts from Ageratum conyzoides for acaricidal properties against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) infesting cattle and buffaloes in India.

In India, Rhipicephalus (Boophilus) microplus populations have developed a certain level of resistance to most of the acaricides marketed against tick species. To manage the problem, the present study was undertaken to evaluate the acaricidal potential of Ageratum conyzoides plants against acaricides-resistant ticks infesting cattle and buffaloes. The regression analysis of dose-response data of ethanolic extract of A. conyzoides revealed LC90 value of 8.91% against reference susceptible IVRI-1 line of R.(B.) microplus. The ethanolic extract was found efficacious against 76.7-90% acaricides-resistant field ticks and adversely affected oviposition showing 7.04-31.3% reduction in egg laying capacity. The extract was also showed an in vitro efficacy of 52.5 and 76.7% against reference resistant IVRI-4 and 5 lines. The GC/MS/MS profiling of hexane extract, two bioactive sub-fractions and essential oils revealed the presence of 6,7-dimethoxy-2,2-dimethyl-2H-1-benzopyran (precocene II) as a major phyto-compound. The bioactive sub-fractions showed 96.2-97.5% efficacy against larvae of IVRI-1 and 77.1-94.9% against multi-acaricide resistant larvae of IVRI-5 line of R.(B.) microplus. The results of this study provided significant support for the development of a phyto-formulation based on A. conyzoides species.

Isolation and characterization of Cu/Zn-superoxide dismutase in Fasciola gigantica.

A full-length complementary DNA (cDNA) encoding Cu/Zn-superoxide dismutase was isolated from Fasciola gigantica that on nucleotide sequencing showed a close homology (98.9%) with Cu/Zn-superoxide dismutase (SOD) of the temperate liver fluke, F. hepatica. Expression of the gene was found in all the three developmental stages of the parasite viz. adult, newly excysted juvenile and metacercaria at transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. F. gigantica Cu/Zn-SOD cDNA was cloned and expressed in Escherichia coli. Enzyme activity of the recombinant protein was determined by nitroblue tetrazolium (NBT)-polyacrylamide gel electrophoresis (PAGE) and this activity was inactivated by hydrogen peroxide but not by sodium azide, indicating that the recombinant protein is Cu/Zn-SOD. The enzyme activity was relatively stable at a broad pH range of pH 4.0-10.0. Native Cu/Zn-superoxide dismutase protein was detected in the somatic extract and excretory-secretory products of the adult F. gigantica by Western blotting. NBT-PAGE showed a single Cu/Zn-SOD present in the somatic extract while three SODs are released ex vivo by the adult parasite. The recombinant superoxide dismutase did not react with the serum from buffaloes infected with F. gigantica. The role of this enzyme in defense by the parasite against the host reactive oxygen species is discussed.

Anthelmintic effect of a methanol extract of leaves of Dregea volubilis on Paramphistomum explanatum.

Dregea volubilis (family Asclepediaceae) is widely used as anthelmintic in traditional system of medicine in eastern and southern part of India. The aim of this study was to evaluate the anthelmintic activity of the methanol extract of D. volubilis leaves (MEDV) and to observe its effect through SEM study. Live parasites (trematode Paramphistomum explanatum) were collected from buffalo in 0.9% phosphate-buffered saline (PBS). It was incubated at 37 ± 1°C in media containing either no extract (control), the test drug, MEDV at four dose levels (10, 25, 50, and 100 mg/ml) or the standard drug, albendazole, at a dose of 10 mg/ml. The effectiveness of the extract was judged on the basis of the loss of spontaneous movement and/or complete destruction or death of the trematodes. After being removed from the experimental medium, trematodes were dipped in PBS at 37 ± 1°C and on gentle stimulation, the paralyzed parasite showed immobility. Death was confirmed when it completely lost its motility even when vigorously shaken or dipped in warm water (50°C). The trematodes, both drug treated and others, were further processed for SEM study using standard method. The anthelmintic activity was found with all the doses through paralysis and death of the organisms (p < 0.001). Maximum anthelmintic activity was found with a dose of 100 mg/ml. Minor damage was observed with both suckers, but severe distortion was found with tegumental surface of the treated trematodes. The present SEM-based study established the anthelmintic activity of MEDV.

Clinical, haematological and therapeutic studies on tropical theileriosis in water buffaloes (Bubalus bubalis) in Egypt.

Thirty buffaloes naturally infected with Theileria annulata and 10 parasitologically free controls were used to determine the potential clinical, haematological and therapeutic impact of tropical theileriosis in Egypt. The clinical signs in the infected buffaloes were pyrexia (40.5-41.5 degrees C), enlargement of superficial lymph nodes, slight nasal and ocular discharges, salivation, anaemia and respiratory distress. Eye lesions also were recorded. There was a significant decrease in erythrocyte counts and haemoglobin content and a significant decrease in total leucocyte counts in infected buffaloes compared to controls. Early treatment with buparvaquone was 100% effective in eliminating the protozoan parasites from the blood and lymph nodes and led to an improvement in the clinical state whereas treatment in the later stages of the disease whilst eliminating the parasites failed to improve the clinical condition of the animal.

Treatment and reinfection of water buffaloes and cattle infected with Schistosoma japonicum in Yangtze River Valley, Anhui province, China.

The aim of the present study was to evaluate the efficacy of praziquantel treatment of Schistosoma japonicum infections in cattle and water buffaloes and to assess the natural rate of reinfection after treatment. The studies were conducted on 2 islands in the Yangtze River, Anhui province, China, from March 2003 to January 2004. The efficacy of praziquantel was 97% when applied orally wrapped in tree leaves at the recommended doses. The efficacy was measured using a miracidium hatching technique on fecal samples collected 20 days after treatment. The treatment did not give rise to any major side effects. Reinfection after treatment was high and occurred throughout the year in both cattle and water buffaloes. Age-related resistance was only observed in water buffaloes. It is concluded that although praziquantel is highly effective against S. japonicum in cattle and water buffaloes, a single annual treatment strategy does not effectively control transmission. New strategies for integrated control of animal schistosomiasis are needed to control schistosomiasis transmission more effectively in farm areas of China.

Some parasitic flukes infecting farm animals in Al-Santa Center, Gharbia Governorate, Egypt.

A total of 213 farm animals in the vicinity of Al-Santa Center (73 cattle, 90 buffaloes and 50 sheep) were coprologic examined for natural infection with trematod-parasites. The results showed that cattle were infected with Fasciola sp (21.8%) and Paramphistomum sp. (7.3%), buffaloes were infected with Fasciola sp. (17.7%) and Paramphistomum sp. (10%), while sheep were infected with Fasciola sp. (30%), D. dendriticum (5%) and Paramphistomum sp. (4%). The three animal species were treated for paramphistomiasis with a total dose of 1800, 6000 and 7500 mgm of Oleo-resin solution of Commiphora molmol (dose of 6 ml of 10gm% equal to 2 Mirazid. The cure was 100% in sheep 80% in cattle and 44.4% in buffaloes. High dose for both cattle and buffaloes to reach 100% cure rate was not tried.

Detection of antibody to Toxocara vitulorum perieneteric fluid antigens (Pe) in the colostrum and serum of buffalo calves and cows by Western blotting.

Toxocara vitulorum, a nematode parasite in the small intestine of cattle and water buffaloes, causes high morbidity and mortality of 1-3 months old buffalo calves. This research evaluated the specific perieneteric antigens (Pe) reactivity of anti-T. vitulorum-Pe antibody (Tv-Pe-Ab) in both immune sera and colostrum from buffalo cows immediately post-partum from buffalo cows. The presence of Tv-Pe-Ab in sera of buffalo newborn calves was also examined at 1 day before and after suckling the colostrum as well as in sera from naturally infected calves at the beginning and peak of the maximum infection and then again during the period of rejection and post-rejection of the parasite. Pe antigens were characterized for Tv-Pe-Ab by SDS-PAGE and Western blot (WB). The SDS-PAGE showed that Pe contained nine protein bands (11, 14, 31, 38, 58, 76, 88, 112 and 165 kDa). All Pe bands were recognized by Tv-Pe-Ab in sera and colostrum of buffalo cows. Only the serum antibodies of buffalo calves at 1 day of age after suckling the colostrum and during the beginning of T. vitulorum infection recognized Pe antigen's nine bands. In contrast, serum antibodies from 1-day-old buffalo calves, taken before suckling colostrum, did not react with any protein band. In suckling calves, which reached peak egg output, rejection and post-rejection stages of the infection, serum Tv-Pe-Ab reactivity with lower molecular weight protein bands (11-76 kDa) was lost and only reactivity with the Pe protein bands of higher molecular weight (88, 112 and 165 kDa) remained.

Antifilarial activity of Azadirachta indica on cattle filarial parasite Setaria cervi.

Alcohol and aqueous extracts of flowers of Azadirachta indica were tested in vitro for their potential antifilarial activity against whole worm, nerve muscle (n.m.) preparation and microfilariae of Setaria cervi. The effects of alcohol and aqueous extracts were similar in nature on the spontaneous movements of whole worm and nerve muscle preparation. On the whole worm, the response was characterized by initial increase in tone, rate and amplitude of contractions followed by reversible paralysis. The initial stimulant effect is likely to be due to irritant effect on the cuticle. Nerve muscle preparation responded to both extracts by inhibition of spontaneous movements followed by reversible paralysis; initial stimulation phase was absent. The inhibition was concentration related. Alcohol and aqueous extracts had almost similar lethal effect on the microfilariae of S. cervi, the LC50 being 15 and 18 ng/ml, respectively.

Humoral immune response of water buffalo monitored with three different antigens of Toxocara vitulorum.

Humoral immune response of water buffalo naturally infected with Toxocara vitulorum was monitored using three different antigens of this parasite in serum and colostrum of buffalo cows and calves. Soluble extract (Ex) and excretory/secretory (ES) larval antigens and perienteric fluid antigen (Pe) of adult T. vitulorum were used to measure the antibody levels by an indirect ELISA. Serum of 7-12 buffalo cows for the first 365 days and colostrum of the same number of buffalo cows for the first 60 days of parturition, and serum of 8-10 buffalo calves for the first 365 days after birth were assayed. The ELISA detected antibodies against all three T. vitulorum antigens in the colostrum and serum of 100% of buffalo cows and calves examined. The highest antibody levels against Ex, ES and Pe antigens were detected in the buffalo cow sera during the perinatal period and were maintained at high levels through 300 days after parturition. On the other hand, colostrum antibody concentrations of all three antigens were highest on the first day post-parturition, but decreased sharply during the first 15 days. Concomitantly to the monitoring of immune response, the parasitic status of the calves was also evaluated. In calves, antibodies passively acquired were at the highest concentrations 24 h after birth and remained at high levels until 45 days coincidentally with the peak of T. vitulorum infection. The rejection of the worms by the calves occurred simultaneously with the decline of antibody levels, which reached their lowest levels between 76 and 150 days. Thereafter, probably because of the presence of adults/larvae stimulation, the calves acquired active immunity and the antibodies started to increase slightly in the serum and plateaued between the days 211 and 365. All three antigens were detected by the serum antibodies of buffalo calves; however, the concentration of anti-Pe antibody was higher than anti-EX and anti-ES, particularly after 90 days of age. By conclusion, the buffalo cows develop immunity and keep high levels of antibodies against T. vitulorum-Ex, ES and Pe antigens and these antibodies are transferred to their calves through the colostrum. This passively acquired immunity does not protect the calves against the acquisition of the infection, but these antibodies, passively or actively acquired, may have an important role during worm rejection by the calves and prevention of intestinal reinfection.

Toxocara vitulorum in the milk of buffalo (Bubalus bubalis) cows.

The duration of excretion of Toxocara vitulorum larvae in the milk of buffalo cows determines the optimum time for treating calves. Studies on 10 cows showed that a few larvae occur in the colostrum of some cows before the calf has suckled, but most are present from the day after calving and for a further five days. From day 9 onwards, very few larvae were found in the milk. The total number of larvae found was comparable with the number of adult parasites collected from the calves of cows with similar histories. The larvae were 1254 +/- 60 microns long and 36 +/- 6.7 microns in diameter at the ventriculus, figures which are substantially different from some published results.