Implementation of an extended ZINB model in the study of low levels of natural gastrointestinal nematode infections in adult sheep.

BACKGROUND: In this study, two traits related with resistance to gastrointestinal nematodes (GIN) were measured in 529 adult sheep: faecal egg count (FEC) and activity of immunoglobulin A in plasma (IgA). In dry years, FEC can be very low in semi-extensive systems, such as the one studied here, which makes identifying animals that are resistant or susceptible to infection a difficult task. A zero inflated negative binomial model (ZINB) model was used to calculate the extent of zero inflation for FEC; the model was extended to include information from the IgA responses. RESULTS: In this dataset, 64% of animals had zero FEC while the ZINB model suggested that 38% of sheep had not been recently infected with GIN. Therefore 26% of sheep were predicted to be infected animals with egg counts that were zero or below the detection limit and likely to be relatively resistant to nematode infection. IgA activities of all animals were then used to decide which of the sheep with zero egg counts had been exposed and which sheep had not been recently exposed. Animals with zero FEC and high IgA activity were considered resistant while animals with zero FEC and low IgA activity were considered as not recently infected. For the animals considered as exposed to the infection, the correlations among the studied traits were estimated, and the influence of these traits on the discrimination between unexposed and infected animals was assessed. CONCLUSIONS: The model presented here improved the detection of infected animals with zero FEC. The correlations calculated here will be useful in the development of a reliable index of GIN resistance that could be of assistance for the study of host resistance in studies based on natural infection, especially in adult sheep, and also the design of breeding programs aimed at increasing resistance to parasites.

Analysis of genetic variability of Fasciola hepatica populations from different geographical locations by ISSR-PCR.

Inter-simple sequence repeats markers were used to determinate the genetic variability of Fasciola hepatica populations recovered from sheep and cattle from Spain (Sp1, Sp2, Sp3 and Sp4), UK (Eng), Ireland (Ir) and Mexico (Mex). Twenty five primers were tested but only five produced 39 reproducible bands, being 71.79% polymorphic bands. This percentage ranged from 10.26% in Sp4 to 48.72% in Sp1, and per host between 28.21 and 48.72% in sheep and between 10.26 and 38.46% in cattle. This relatively low range of genetic diversity within populations, with a mean of 34.40%, implies that a large proportion of variation resided among populations. The population differentiation (Gst = 0.547) indicated that 54.7% of variation is due to differences between populations and 45.3% due to differences within population. The Nei's distance ranged between 0.091 and 0.230 in sheep and between 0.150 and 0.337 in cattle. The genetic relationships between populations and individuals were shown by a UPGMA dendrogram and a principal coordinate analysis; both grouped all populations separately from Sp4, a population of from the Midwest of Spain with the lowest level of diversity. Small genetic distances were observed between Eng and Ir, on the one hand, and Sp1, Sp2, Sp3, from the Northwest of Spain, together with Mex, on the other.

Efficacy of an anthelmintic combination in sheep infected with Fasciola hepatica resistant to albendazole and clorsulon.

In the current study, we identified five sheep flocks with fasciolosis in the province of León (northwestern Spain) in order to determine the anthelmintic resistance status to three commonly used anthelmintics, namely albendazole (ABZ), triclabendazole (TCBZ) and clorsulon (CLOR). The identification of one flock resistant to ABZ and CLOR was shown after the faecal egg count reduction test (FECRT). The reductions in eggs per gram values were -17.6% and -68% against immature and adult flukes, respectively, after ABZ treatment; 85.15% and 44.91% against immature and adult flukes, respectively, after CLOR treatment; and 97.06% against both stages, after the administration of TCBZ. As an alternative to control the infection, two combinations of ABZ and CLOR were tested. In the first, both drugs were administered at the recommended dose of each; in this case, the efficiency reached values above 95% against both immature and adult flukes. However, when the combined drugs were administered at half the recommended dose of each, the efficiency of the combination was very low, i.e. 16.67% and -11.11% against mature and immature flukes, respectively. In conclusion, this preliminary report suggests a possible interaction between ABZ and CLOR after their joint administration. However, these results should be confirmed in other flocks.

Effect of lipopolysaccharide (LPS) from Ochrobactrum intermedium on sheep experimentally infected with Fasciola hepatica.

The effects of the lipopolysaccharide (LPS) from Ochrobactrum intermedium was evaluated in sheep experimentally infected with Fasciola hepatica. Animals were divided into four groups: two treated with the LPS (T1/T2) and two controls (C1/C2). T1/C1 were slaughtered at 30 days postinfection (dpi) and T2/C2 at 85 dpi. Body weight and body condition were found higher in T1 and T2 than in controls, although differences were not significant. Treated sheep showed lower cumulative fecal egg count than controls (p < 0.01). Levels of red blood cells (RBC), white blood cells, hemoglobin, and hematocrit (HCT) were higher in T1 and T2. Significant differences (p < 0.05) in RBC and HCT were found between groups at 84 dpi. More severe macrocytic and hypochromic anemia was observed in C1 and C2 than in treated groups. Total protein and albumin values were higher in T1 and T2 (p < 0.01) until 29 dpi. At the end of the trial, no significant differences were observed in hepatic enzymes, although gamma-glutamyl transferase and aspartate aminotransferase values were higher in C2, and alanine aminotransferase was higher in T2. At necropsy, the mean weight of liver, fibrosis in portal triads, and ganglion size were similar in all groups. The number and size of flukes was greater in C2 than in T2 (p < 0.05). The histological examinations revealed a higher degree of parenchymatous fibrosis in T2 compared to C2 (p < 0.05). The administration of LPS from O. intermedium increased the nonspecific resistance against F. hepatica in experimentally infected sheep.

Characterization of a multidrug resistant Teladorsagia circumcincta isolate from Spain.

The aim of this work was to know the anthelmintic resistance (AR) status of a Spanish sheep flock infected by gastrointestinal nematodes (GIN) and the possible cross resistance among anthelmintics of the macrocyclic lactones (ML) family. The Faecal Egg Count Reduction Test (FECRT) was carried out to check the efficacy of albendazole (Zodalben®), levamisole (LEV) (Endex®) and an oral formulation of ivermectin (IVM) (Oramec®), at the recommended dose rates. Then, the study was extended to check the cross resistance between drugs of the ML family: injectable IVM (Ivomec®), oral moxidectin (Cydectin®), injectable moxidectin (Biodectin®) and doramectin (Dectomax®), at the recommended dose rates. The GIN species were identified after faecal cultures in all groups. The FECRT showed the resistance of a Teladorsagia circumcincta isolate against LEV (39-58%), IVM (88-92%) and doramectin (85%). This study is the first report to confirm the side resistance between these MLs, which belong to the avermectin chemical group, in a Spanish sheep flock. The in vitro efficacy of LEV and IVM was measured by the Larval Feeding Inhibition Assay (LFIA) using the IC(50) measurement (concentration needed to inhibit the ingestion of 50% L1). The values of the multidrug resistant isolate were 0.25 µg/ml for LEV and 3 ng/ml for IVM. Both results were higher than the values obtained with a susceptible isolate, which could be indicative of AR. However, further research examining the response of a greater range of susceptible and resistant nematodes isolates should be carried out to establish a discrimination threshold.

Immunization protocols against Cryptosporidium parvum in ovines: protection in suckling lambs.

Ovine colostrum and milk from immunized ewes were tested for their ability to prevent cryptosporidiosis in the lambs experimentally infected with 10(6) oocysts of Cryptosporidium parvum at 36-48 h of age (day 0 post-infection). All lambs became infected and developed clinical cryptosporidiosis. However, lambs fed by immunized dams have shown shedding involved, significantly, fewer oocysts and lasted for a shorter period than in control lambs. In addition, diarrhoea was less severe. The best results emerged in lambs of ewes immunized by intramuscular injection of an emulsion of 2 ml of Freund's complete adjuvant and 2 ml of C. parvum antigen in sterile phosphate buffered saline solution, administrated four weeks before parturition, together with an intramammary infusion of 25 microg of antigen in 2 ml of sterile PBS emulsified in 2 ml of Freund's incomplete adjuvant, which showed the highest anti-C. parvum titres in lacteal secretions. In their case, the onset of output of oocysts was delayed by two days, the patent period was shortened by three days, their diarrhoea continued for only three days, and the quantity of oocysts shed decreased by 77%. The outcome was that at the end of the study they had a live weight gain of 2 kg more than the lambs in the control group. These results indicate that lactogenic immunoprophylaxis should help mitigate the financial losses caused by cryptosporidiosis in small ruminants, as well as reducing the risk of infection of humans through the decreased contamination of the environment with oocysts.

Real time PCR for the diagnosis of benzimidazole resistance in trichostrongylids of sheep.

This report describes a new molecular method for the diagnosis of benzimidazole susceptibility or resistance in three main species of trichostrongylids of sheep (Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus vitrinus). This assay is based on the use of real time polymerase chain reaction (PCR) to detect mutations of residue 200 on isotype 1 of beta-tubulin. The technique allows calculation of the proportion of each allelic variant as it combines kinetic (real time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The level of resistance in the population is determined by the proportion of the beta-tubulin codon 200 TAC allele. Hence, it was observed that the proportion of the resistant allele in susceptible strains ranged between 24% and 32.3%; in resistant strains this value increased to between 71.3% and 86.3%. Furthermore, there was a good correlation between real time PCR, faecal egg count reduction test, egg hatch assay and conventional allele-specific PCR, in both resistant and susceptible strains. A sensitive, rapid, highly reproducible and inexpensive technique for detecting resistance to benzimidazoles in a worm population has been developed.

Oral administration of hyperimmune anti-Cryptosporidium parvum ovine colostral whey confers a high level of protection against cryptosporidiosis in newborn NMRI mice.

Hyperimmune anti-Cryptosporidium parvum ovine colostral whey (HOCW) was tested to determine whether it conferred passive immunity to newborn NMRI mice. Three HOCWs (groups IV-VI), 2 nonimmune colostral wheys (groups II and III), and PBS (group I) were administered once (experiment A) and 3 times (experiment B) daily from -1 to 15 days postinfection (PI). Mice in groups I-VI were inoculated with 5 x 10(5) oocysts (day 0 PI), and group VII mice acted as controls. The percentage and intensity of infection were measured at 6, 9, 12, and 16 days PI. In experiment A, HOCW did not reduce significantly the percentage and intensity of infection except for mice in group VI treated with HOCW with the highest titers of anti-C. parvum antibodies. In contrast, no infection was detected in between 18.7 and 62.5% of the mice in groups IV-VI in experiment B. Furthermore, in these groups, the intensity of the infection decreased significantly, ranging from 83.5 to 97.4%. Thus, HOCW did not completely avoid infection, but a high level of protection was observed, being proportional to the titer of specific antibodies and the amount of whey administered orally. Finally, group VII showed no presence of oocysts.

Resistance of gastrointestinal nematodes to the most commonly used anthelmintics in sheep, cattle and horses in Spain.

The objective of this study was to evaluate the status of anthelmintic resistance (AR) in ruminants and horses in Spain. The efficacy of commonly used macrocyclic lactones (MLs) - ivermectin (IVM) and moxidectin (MOX) - was measured in sheep, cattle and horses. In addition, albendazole (ABZ) and levamisole (LEV) were evaluated in sheep and oxibendazole (OXI) and pyrantel (PYR) in horses. Efficacy was evaluated based on the difference between the arithmetic mean pre- and post-treatment faecal egg count (in cattle and horses), or compared to an untreated control group (in sheep). AR was present when the percentage reduction in egg count was <95% and the lower 95% confidence interval (CI) was <90%; if only one of these two criteria was met, the finding was recorded as suspected AR (SAR). In horses, AR-PYR and OXI was considered when the percentage reduction in egg count was ≤ 90% and the lower 95% CI was ≤ 80%. For each animal species, at least 10 study sites were selected. AR to at least one of the drugs was detected in all 10 sheep flocks; the main parasite identified after treatment was Teladorsagia circumcincta. Moreover, in 5 flocks multidrug resistance was identified, on 4 farms to drugs from different families, on one farm to both MOX and IVM and on another farm to all drugs tested. In cattle, the efficacy of both MOX and IVM was 100% on 4 and 3 farms, respectively, and therefore 60% of these farms were considered to have AR or SAR to both MLs. The most frequent parasite identified after treatment was Trichostrongylus spp., although Ostertagia ostertagi was also identified after treatment on one farm. In contrast to ruminants, the 4 drugs evaluated in horses were highly efficacious against strongyles, with efficacies for the MLs and OXI between 95 and 100% and between 94 and 100% for PYR, although 3 herds were SAR against PYR. In conclusion, AR to at least one of the commonly used drugs was identified on all sheep flocks investigated in the northwest of Spain. The occurrence of AR to MLs in cattle was higher than expected but consistent with what was observed in sheep. In horses, all currently used drugs were confirmed as effective against strongyles.

Survival and infectivity of Dicrocoelium dendriticum eggs under field conditions in nw Spain.

A study was made of the survival of Dicrocoelium dendriticum eggs in sheep faeces in an area with a temperate climate (NW Spain). It appears that mortality is independent of the age of the eggs and that there is a marked seasonality within the period of time considered (20 months). A study was also carried out on the infectivity of D. dendriticum eggs by experimental infections of suitable intermediate hosts. The data show that no loss took place in infectivity during the period of study (15 months).

The overwintering of eggs, intramolluscal stages and metacercariae of Fasciola hepatica under the temperatures of a Mediterranean area (Madrid, Spain).

The survival of embryonated and unembryonated eggs, of snails with mature or immature infection and of metacercariae over the winter of a Mediterranean area was analyzed. Embryonated eggs were more resistant than unembryonated eggs to cold weather, leaving a residual contamination which was responsible for earlier spring infection of the snail. Overwintering was similar in snail populations with both mature and immature infections although the lifespan was shorter in the former. Both snail populations survived in the mildest winter but not in the coldest. Only metacercariae from mid autumn were able to overwinter in an significant proportion (45%) but they were non-viable by mid spring. Our results suggest that in very cold winters no risk for grazing animals should be expected in spring.

The effect of the host's age on the pathology of Eimeria stiedai infection in rabbits.

The pathology of experimental Eimeria stiedai infection in 2- and 4-month-old rabbits raised coccidia-free was studied. Over 50 days of infection, the serum activities of aspartate (AST) and alanine (ALT) aminotransferases, alkaline phosphatase (AP) and gamma-glutamyltranspeptidase (GGTP) were analyzed as indicators of hepatic lesions and total serum globulins with respect to the immune response of the host; parasite development was followed by oocyst output; weight gain, food intake and relative liver weight were analyzed to control the performance of infected animals. The age of the host strongly affected parasite development and consequently the biochemical and zootechnical parameters differed more markedly in the younger animals; however, the enzyme ALT showed an increase independent of parasite development, and infection did not affect GGTP activity. The increase in age was responsible for resistance of rabbits to hepatic coccidiosis and older animals were less affected than young ones.

Efficacy of moxidectin 1% injectable and 0.2% oral drench against natural infection by Dictyocaulus filaria in sheep.

Two separate trials (I and II) with 34 and 32 Churra ewes, respectively, and distributed into two groups, have been carried out to evaluate the efficacy of two different formulations of moxidectin at a dose rate of 0.2mg/kg body weight (b.w.) against natural infection by Dictyocaulus filaria in sheep. Trial I was designed to evaluate a 1% moxidectin injectable formulation, whereas in trial II a 0.2% moxidectin oral drench formulation was used. The efficacy was measured on the basis of the reduction of the faecal larval counts and of adult worm recoveries at slaughter. In each trial, a group of animals was treated on day 0 with moxidectin 1% injectable or moxidectin 0.2% oral drench and the other group acted as untreated control. When the faecal larval counts was compared within the treated groups, the efficacy was over 95% until day +13, and 100% at the remainder of the sampling dates after the application of injectable moxidectin, whereas in trial II, the larvae per gram (lpg) of faeces increased until the first sampling time post treatment (p.t.), day +6, and zero counts were recorded for all animals by the following days. On the basis of adult worm recoveries at necropsy, the efficacy of the treatment was 100% in both trials, however, adult worms were detected at slaughter for all control sheep. These results indicate that moxidectin 1% injectable and moxidectin 0.2% oral drench, administered at 0.2mg/kg b.w., were 100% effective against D. filaria infection in sheep. No adverse reactions to the treatments were observed in the animals.

The acid phosphatase activity and morphological characteristics of Dipetalonema dracunculoides (Cobbold, 1870) microfilariae.

The acid phosphatase activity and some morphological characteristics of Dipetalonema dracunculoides microfilariae are described. Their morphological features are closely related to those of the pathogenic Dirofilaria immitis when Knott's technique is used for the microfilarial diagnosis. The acid phosphatase activity pattern found in Dip. dracunculoides microfilariae is clearly different from those previously described for D. immitis, D. repens and Dip. reconditum.

Serum and bile antibody responses (IgG and IgA) during subclinical Fasciola hepatica infection in sheep.

The kinetics of serum and bile immunoglobulins (IgG and IgA) directed against Fasciola hepatica in the course of subclinical infection induced experimentally was investigated in sheep. Serum activities of glutamate dehydrogenase and gamma-glutamyltransferase were used as markers of the different fluke stages during infection and associated liver damage. Specific serum and bile immunoglobulins followed a similar kinetic pattern, increasing progressively from infection throughout the prepatent period and tended to decrease when adult flukes became established in the bile duct. IgA titres were lower than those of IgG. Specific IgG and IgA bile titres reached maximum values at 14 weeks postinfection that were considerably lower than the serum titres during the whole experimental period. The major bile immunoglobulins are probably derived directly from plasma. The immunoglobulin kinetic pattern could be related to changes in serum liver enzyme activities.

Experimental ovine fasciolosis: antipyrine clearance as indicator of liver damage.

Antipyrine clearance was used to assess microsomal oxidative function in eight female Churra breed sheep at 20, 30, 40, 60, 80 and 100 days after infection by an oral administration of 150 metacercariae of Fasciola hepatica. Experimental infection was ascertained by an ELISA test and by faecal analysis. A significant increase in plasma glutamate dehydrogenase (GLDH) activity from 20 days post-infection and in gamma-glutamyltransferase (GGT) activity from 40 days post-infection was found. Both enzyme activities reached maximum levels in plasma of infected sheep at 80 days post-infection, progressively decreasing thereafter. A significant reduction in the total plasma clearance of antipyrine occurred from 60 to 100 days post-infection and a significant increase in mean residence time occurred by 80 days post-infection. The decrease of antipyrine metabolism coincided with the entrance of parasites in bile ducts and the highest liver damage caused by migrating juvenile flukes.

Experimental transmission of Dipetalonema dracunculoides (Cobbold 1870) by Rhipicephalus sanguineus (Latreille 1806).

A dog naturally infected with Dipetalonema dracunculoides and having a microfilaremia of 6050 microfilariae per mm3 of blood was used as source of infection. Experimentally cultivated nymphs of Rhipicephalus sanguineus were fed on the donor dog. Once engorged, ninety-three nymphs were removed and kept at 30 degrees C and RH 90% until they moulted to the adult stage. To study the development of microfilariae in the vector, ten ticks were dissected at Day 37 post-infection. Adult infected ticks were fed on two uninfected dogs. The observed pre-patent periods were 69 and 76 days. Trans-stadial transmission of Dipetalonema dracunculoides by Rhipicephalus sanguineus was demonstrated.

Serum antibody response in lambs naturally and experimentally infected with Cryptosporidium parvum.

The immunoglobulin (IgG), IgM and IgA responses in Castellana-Manchega cross-bred colostrum-deprived and colostrum-fed lambs infected neonatally with Cryptosporidium parvum were measured using an enzyme-linked immunosorbent assay. A comparison of oocyst shedding and anti-C. parvum serum IgG levels in lambs suffering either natural or experimental infection was undertaken. Effects on the oocyst shedding and IgG levels of C. parvum rechallenge at 30 and 120 days of age in neonatally infected lambs were also evaluated. Anti-C. parvum immunoglobulin levels in colostrum-deprived animals peaked on Day 30 of life for IgG and on Day 15 for IgM and IgA. Lambs that received maternal colostrum showed elevated anti-C. parvum IgG, IgM and IgA levels at 3 days old indicating a transfer of colostral immunoglobulins. Experimentally infected lambs showed a IgG response similar to naturally infected lambs, suggesting that the serum IgG response is independent of the infective dose. Finally, lambs rechallenged at 30 and 120 days old did not show either appreciable oocyst shedding or any increase in their anti-C. parvum IgG levels when compared with prechallenged animals.

Appetite depression in sheep experimentally infected with Fasciola hepatica L.

The purpose of this study was to determine the effects of experimental fasciolosis at various stages of development on the daily food intake of sheep. Five male Churra sheep, 4 months of age, were infected orally with 300 Fasciola hepatica metacercariae over a 30 day period. There was a significant increase in serum glutamate dehydrogenase (GLDH) activity from 40 days post-infection and in aspartate aminotransferase (AST) activity from 60 days post-infection. Both enzyme activities reached maximum levels in the serum of infected animals at 80 days and then progressively decreased. Serum gamma-glutamyltransferase (GGT) activity was significantly increased from 80 to 120 days post-infection. Glycaemia was significantly decreased from 60 days post-infection. The average daily food intake was shown to steadily decrease until approximately 100 days. The coincidence of decreased food intake with the period of significant increase, both in AST and GLDH activities, indicated that damage caused around the time of migration of immature flukes through the liver parenchyma may be involved in appetite depression.

Efficacy of moxidectin 1% injectable against natural infection of Sarcoptes scabiei in sheep.

Thirty ewes naturally infected with Sarcoptes scabiei var. ovis, were allocated into three groups of 10 animals each. Animals in groups B and C were treated on day 0 and on days 0 and +10, respectively, with moxidectin 1% injectable at a dose of 0.2mg moxidectin/kg body weight (BW). Group A remained untreated. Seven days before treatment, the geometric mean of Sarcoptes scabiei var. ovis per square centimeter of skin in groups A, B and C were not significantly different. From the day of treatment to the end of the trial, the average number of mites/cm(2) increased in untreated animals and decreased in groups B and C, but these values were higher for group C. Active lesions produced by S. scabiei var. ovis consistently increased during the trial in the untreated animals; in group B the minimum count occurred on day +56 this reduction being more evident in group C (no lesions on days +49 and +56). Also in this group, the number of cured animals was 100%, therefore, the application of two treatments with moxidectin (group C) showed higher efficacy than a single treatment (group B). Body condition score decreased in the three experimental groups along the trial. All animals were individually weighed on days -1, +28 and at the end of the trial. No adverse reactions were observed in the animals treated with 0.2mg moxidectin/kg BW.