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.

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.

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.

Immunological features of LPS from Ochrobactrum intermedium on sheep experimentally infected with Fasciola hepatica.

The effects of the lipopolysaccharide (LPS) from Ochrobactrum intermedium in sheep with fasciolosis was reported previously, resulting in lower fecal egg counts and fluke burden. In the current study, we analyzed its immunological effects in two groups of sheep, treated (T) and controls (C). Fasciolosis induces a T helper (Th) type-2 response, characterized by IL-4 and IL-10 production; however, at the beginning of the infection, the IFN-γ production predominates (Th type-1 response). Although we did not find differences in IL-4 production or in the expression level of this gene in the hepatic lymph nodes, the expression level of IL-10 was higher (P < 0.05) in the T group at 4 wpi. The IFN-γ production was higher (P < 0.01) at 12 wpi as well as its level of expression at 4 wpi (P < 0.05) in the T group. We found a higher expression level of TGF-ß at 4 wpi in the T group (P < 0.05), associated with the previous report of thicker fibrous tracks in a treated group. Immunoglobulin G1, related with a Th type-2 response, was higher (P < 0.01) in the T group at 4 and 12 wpi. In conclusion, the effects of LPS from O. intermedium could have resulted from a predominant Th type-2 immune response.

Effect of dietary supplementation with flaxseed oil or vitamin E on sheep experimentally infected with Fasciola hepatica.

The effects of two diets supplemented with flaxseed oil (FO) or vitamin E (VE) were studied in sheep infected with Fasciola hepatica. The control group (CO), not supplemented, had higher weight gain and faecal egg count (P < 0.05) at the end of the study. At necropsy, the number (71.2 ± 26.5) and size of flukes were lower in the FO group (P < 0.01), probably due to the higher levels of white blood cells and lymphocytes (P < 0.01). This group also had higher red blood cell and haematocrit values (P < 0.01). The CO group had more severe hypoproteinaemia and hypoalbuminaemia, due to having the highest fluke burden (123.0 ± 35.2), and also the highest IgG1 titre (P < 0.01). The diet did not affect production or gene expression levels of IFN-γ and IL-4. On the other hand, the supplementation with VE led to a reduction in adult fluke burden (97.7 ± 39.9) and lower lipid oxidation in the liver (P < 0.001).

Development of an egg hatch assay for the detection of anthelmintic resistance to albendazole in Fasciola hepatica isolated from sheep.

The aim of this study was to develop an egg hatch assay (EHA) to detect the resistance of Fasciola hepatica to albendazole (ABZ). With this purpose, two different F. hepatica isolates were tested: (i) susceptible (Shrewsbury/South Gloucester strain) to ABZ (SA); (ii) resistant to ABZ (RA). A commercial formulation of ABZ (Sinvermin) diluted in dimethyl sulfoxide (DMSO) was used at concentrations of 0.0002, 0.002, 0.02, 0.2, and 2 µg/ml. In the SA isolates, eggs from feces exposed to ABZ at the three highest concentrations showed significantly lower hatching percentages than the control well (P<0.01); also for the two highest concentrations, the percentage of development was significantly lower than the control (P<0.01). However, in the RA isolate, no differences among treatments and control were found in the percentage of hatched or developed eggs. On the other hand, the EHA was conducted over a 15 day period following treatment with ABZ for sheep infected by the RA isolate. On day 7 post-treatment (pt), no differences among treatments and controls were observed. On day 15 pt, the development and hatching rates exposed to the highest ABZ concentration were lower than at lower concentrations, suggesting susceptibility. The ovicidal effect of ABZ and the potential use of EHA to differentiate between ABZ susceptible and resistant isolates are confirmed in the current study. However, the use of this technique should be confirmed with a higher number of isolates.

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.

The diagnosis of fasciolosis in feces of sheep by means of a PCR and its application in the detection of anthelmintic resistance in sheep flocks naturally infected.

The aim of this study was to develop a PCR for the diagnosis of Fasciola hepatica infection in feces of sheep based on the ribosomal internal transcribed spacer. Detection of infection was possible from the second week post-infection in experimentally infected sheep by amplification of a 292bp fragment. This PCR was employed for the detection of anthelmintic resistance (AR) in naturally infected sheep flocks, and results were compared with techniques such as the fecal egg count reduction test (FECRT) and the copro-antigen reduction test (CRT). The FECRT was carried out in two flocks, Santillan de la Vega (SV) and Corullón (CR), with sheep treated with albendazole (ABZ), clorsulon (CL), or triclabendazole (TCBZ). Feces were collected from individuals on days 0, 7, 15, and 30 post-treatment (pt). The FECRT showed adult F. hepatica to be resistant to ABZ and CL in both flocks. All parasite stages in the SV flock were susceptible to TCBZ, while in the CR flock, adult flukes showed resistance and immature forms were susceptible to the treatment. To compare FECRT and the PCR results, we calculated the percent of positive sheep on day 1 pt. In both flocks, the percent positive sheep was consistently higher by PCR than by sedimentation, confirming that the PCR is a more sensitive method of diagnosing infection and therefore to detect the resistance in infected animals. The CRT was carried out in the SV flock using a sandwich ELISA kit. The percent of sheep found positive by PCR was higher than with ELISA. Comparison of FECRT, CRT, and PCR for the detection of AR showed PCR to be the most sensitive.

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.

Efficacy of moxidectin long-acting injectable formulation (1 mg/kg bodyweight) against first instar larvae of Oestrus ovis in naturally infected sheep.

The objective of the current study was to evaluate the efficacy of a single treatment with a long-acting injectable formulation of moxidectin (MOX) at 1.0 mg/kg bodyweight (b.w.) against natural infection by nasal bots (Oestrus ovis) in sheep with special attention to first instar larvae (L1). Firstly, a local farm with clinical history of oestrosis was chosen to conduct the assay. A total of 49 sheep were pre-selected at the end of the summer according to the presence of evident clinical signs of infection and confirmed later by means of an indirect ELISA against excretory-secretory products from L1 to detect IgG antibodies. After that, 24 sheep were chosen to carry out the study on the basis of positive serology and age since the oldest ones were selected. The day 0 of the assay, the treatment group was administered with the MOX formulation by subcutaneous injection at the base of the left ear and the control group was administered with a saline solution in the same way. All sheep were slaughtered on day 28 post-treatment (pt). At the necropsy, the head of all sheep were cut off and split into two sagital sections and all larvae from nasal passages, septum, middle meatus, conchae and sinuses were recovered. After the necropsy, a significant number of L1 was only found in the control group and therefore the efficacy of the MOX formulation was only calculated against this stage. As a result, the formulation was 90.2% effective against L1 for sheep slaughtered at day 28 pt.

The present status of anthelmintic resistance in gastrointestinal nematode infections of sheep in the northwest of Spain by in vivo and in vitro techniques.

The aim of this study was to update the anthelmintic resistance (AR) status in sheep flocks infected by gastrointestinal nematodes (GIN) by means of in vivo and in vitro methods in the northwest of Spain. With this objective, we studied the efficacy of benzimidazoles (BZs), imidazothiazoles (IMs) and macrocyclic lactones (MLs), between 2006 and 2011. The sampling area was the Autonomous Community of Castilla y León but the majority of the flocks were located in the province of León. When the mean of GIN eggs per gram (epg) in faeces in a flock was higher than 150, the in vivo Faecal Egg Count Reduction Test (FECRT) was carried out. According to this test, AR was present in 63.6% of flocks, independently of the anthelmintic used. Flocks were mainly resistant to levamisole (LEV) (59.0%), followed by ivermectin (IVM) (27.3%) and albendazole (13.6%). Multidrug-resistance was also observed in 27.2% of the flocks, one of them being resistant to all anthelmintic families, including long-acting moxidectin. Comparing the evolution of AR in the last decade, between 1999 and 2011, the level of resistance to BZs and MLs was fairly constant throughout the time by means of the FECRT. However, the resistance to LEV increased significantly in only one decade since during the period 1999-2003 the percentage was 38.5%. The AR status was also measured by in vitro techniques in those flocks with an egg output lower than 150 epg. The prevalence of AR to BZs reached the 35.3% by Egg Hatch Assay. However, the level of resistance reported for LEV and IVM was 61.5% and 23.5%, respectively, by using the Larval Feeding Inhibition Assay, percentages very similar to those reported with the FECRT.

Comparison of three different techniques to diagnose Fasciola hepatica infection in experimentally and naturally infected sheep.

The aim of this study was to compare three different techniques for the early diagnosis of the infection by Fasciola hepatica in experimentally and naturally infected sheep. The experimental group consisted of 7 sheep infected with 200 metacercariae; faecal samples were taken weekly until 12 week post-infection (wpi). Under natural conditions, 45 individual faecal samples and 2 pools of faeces were collected from three different flocks with a history of F. hepatica infection. The results obtained by a coprological method were compared with a commercial immunoassay and with two PCR assays in faecal samples. Faecal eggs were detected by 9 wpi in experimental infection. On the other hand, only 24 out of 45 sheep were positive in naturally infected flocks. By means of a sandwich-ELISA kit, the infection was first detected by 4 wpi in the 57.1% experimentally infected sheep and this percentage reached 100% by 8 wpi. All naturally infected animals were positive with this method. Regarding PCR, a specific 423 bp fragment of mitochondrial DNA was amplified in faecal samples. The F. hepatica infection was detected from 3 wpi with a standard PCR, and from 2 wpi with a nested-PCR. Only 37 sheep out of 45 were positive by the standard PCR although the infection was diagnosed in all animals by the nested-PCR. In conclusion, the sensitivity of the nested-PCR described in our study is higher than the detection of eggs in faeces as well as the commercial immunoassay. Moreover, no cross reactions were described with gastrointestinal nematodes.

Teladorsagia circumcincta antibodies in serum and milk samples in experimentally infected lactating ewes.

The aim of this study was to evaluate the IgG activity in serum and milk samples of experimentally infected lactating ewes with the nematode Teladorsagia circumcincta as well as its relationship to the degree of infection. In a previous study 28 pregnant ewes were divided into two homogeneous groups, with high (H) and low (L) level of parasitism, respectively. Blood and milk samples were taken weekly after lambing until the end of the lactation, 126 days post-partum (pp). IgG against T. circumcincta were measured by means of an indirect ELISA. The kinetic of the immunoglubulins in serum samples showed a very low activity at the beginning but gradually increased throughout the lactation; H group showed higher values most of the sampled days than L group. Contrary, IgG in milk samples remained high during the first month pp, then decreased around 38% and by the end of the study rose again. Antibodies in both samples were correlated and especially during the second month of lactation (r=0.3; p<0.001). With the aim to correlate the immune response and the degree of infection we found an inverse relationship (r=-0.2; p<0.05) during the second month of lactation between eggs and IgG in serum. However the correlation with immunoglobulins in milk was positive, mainly, on the last third of lactation (r=0.2; p<0.01). As a conclusion, the individual detection of total IgG antibodies against T. circumcincta in lactating ewes is highly dependent on the stage of lactation. Therefore, these associations should be confirmed under field conditions.

Update on trematode infections in sheep.

Trematode parasites live in the liver, fore stomachs or blood vessels of a wide range of animals and humans. Most of them have a special economic and veterinary significance. Liver fluke disease of sheep and other animal species is caused by the common liver fluke Fasciola hepatica. Hepatic fasciolosis occurs throughout the world, where climatic conditions are suitable for the survival of aquatic intermediate host snails. Also of importance for ruminants, in some parts of the world, are Fasciola gigantica and Fascioloides magna. Other trematodes infecting ruminants include Dicrocoelium dendriticum; Eurytrema pancreaticum and Eurytrema coelomaticum. Among the Paramphistomidae, some species can infect sheep and other ruminants. Finally, Schistosoma spp. are found in the blood vessels of ruminants and are of minor importance in temperate regions. The manuscript concentrates on trematode species of veterinary importance for domestic 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.

Effect of infection with Teladorsagia circumcincta on milk production and composition in Assaf dairy sheep.

Infection by gastrointestinal nematodes (GIN) in grazing sheep can negatively influence the growth of lambs and milk yield. In the current study we evaluated the effect of high and low levels of infection with Teladorsagia circumcincta on production parameters of lactating ewes. Two groups of ewes were infected with 50,000 T. circumcincta third stage larvae (L3) 30 days before lambing. To obtain a high level of infection, Group HI was challenged with 30,000 L3 30 days post-partum (pp) and again 90 days pp with the same dose. The low infection group (LI) was treated with an intraruminal controlled-release albendazole bolus one-month pp. We determined the effect of the differing parasite loads on milk yield, milk composition, weight gain, and body condition (BC). Throughout the trial the milk yield was higher in the LI group and, consequently, at the end of the lactation, this group produced 11.1% more milk than the HI group. In the final third of lactation there was an overall increase in milk fat, protein, and lactose although the factor most affected by infection level was the protein percentage, with an increment of 11.9% in the HI group. The somatic cell counts remained low during the experiment indicating the absence of udder infection. Regarding weight and BC, significant differences were found only in BC although LI ewes weighed 2% more than the HI group. In conclusion, a low level of GIN infection in sheep leads to a better BC and a higher milk production with high protein percentage.

Efficacy of a moxidectin/triclabendazole oral formulation against mixed infections of Fasciola hepatica and gastrointestinal nematodes in sheep.

We have evaluated the efficacy in sheep of a combination drench formulation at the recommended dose rate of 0.2 mg moxidectin/kg bodyweight and 10 mg triclabendazole/kg bodyweight against an experimental infection with Fasciola hepatica and a natural infection with gastrointestinal nematodes. We confirmed that the efficacy of reducing fecal egg output was 98.3% for trichostrongyle eggs and 100% for F. hepatica eggs. Based on adult worm and fluke recovery, the efficacy varied according to the target species. A reduction was found in the number of Teladorsagia circumcincta, Trichostrongylus spp., Nematodirus spp., and Trichuris spp. greater than 95%, but the efficacy for Oesophagostomum spp. varied, with values below 90%. The reduction in F. hepatica was higher than 95% for all stages. The effectiveness of the formulation was also confirmed by an increase in total proteins and albumin following treatment.

Cross-sectional sero-epidemiological survey of sarcoptic mange in sheep of Western Castile and Leon, Spain.

The aim of the present study was to determine the prevalence of Sarcoptes scabiei-infection of ovine livestock in three provinces (Leon, Zamora and Salamanca) in the Western part of the Castile and Leon region in Spain, and to determine the association between different variables and seropositivity. A total of 3730 sheep sera from 373 flocks (10 sera from each flock) collected from May to September over the course of the years 2006 and 2007 were individually analysed by an indirect antibody ELISA validated for diagnosing sarcoptic mange in sheep. The overall flock-level true prevalence was 22.6% (95% CI: 17.8-27.4), the overall individual-level true prevalence within the total flocks was 7.2% (95% CI: 6.1-8.3) and the overall individual-level true prevalence within the seropositive flocks was 31.3% (95% CI: 27.2-35.4). The apparent prevalences, at flock-level and at individual-level within the total flocks and within the seropositive flocks, were not statistically different (p > 0.05) when the primary production objective of the flock is milk vs. meat, or in smaller (< or = 276 sheep, 50th percentile) vs. larger flocks (> 276 sheep). The apparent prevalences, at flock-level and at individual-level within the seropositive flocks, were, likewise, not statistically different between the three provinces, but the individual-level apparent prevalence within the total flocks showed significant variation from one province to another (p < or = 0.05). Sheep maintained in the Provinces of Zamora and Salamanca had greater odds (OR = 1.7, 95% CI: 1.2-2.6; OR = 1.9, 95% CI: 1.3-2.8, respectively) of being seropositive than those located in Leon Province (OR = 1.0). The findings of the present study clearly show the need to implement in this region effective control measures against sarcoptic mange in sheep.

Development and evaluation of an antibody ELISA for sarcoptic mange in sheep and a comparison with the skin-scraping method.

In this work an indirect ELISA for detecting serum-specific IgG antibodies in sheep was developed using a crude saline extract from Sarcoptes scabiei var. ovis mites and then the repeatability of the ELISA outcomes was estimated. Subsequently, its diagnostic accuracy was evaluated by Receiver Operating Characteristics (ROC) analysis using a sample collected from the entire sheep population of western Castile and Leon region in Spain, and then compared with that of the skin-scraping method. The reference method used was a combination of clinical examination, skin-scraping analysis and epidemiological surveys, but it introduced selection and probably information biases. Furthermore, we attempted to identify biological factors useful to predict the sensitivity or specificity of the ELISA as determined by comparison with the reference method. Additionally, conventional latent-class analysis [Hui, S.L., Walter, S.D., 1980. Estimating the error rates of diagnostic tests. Biometrics 36, 167-171] was also used to estimate accuracy parameters. The between-run coefficient of variation (CV) for a standard serum was 8.8% and the within-run CV 4.3%. No significant deviation between the OD% means and strength positive correlation between the OD% values (r=0.98) were found for the results from two different batches of antigen. When compared to the reference method, the Area Under the ROC curve (AUC) for the reference population was 0.967 (95% CI: 0.949-0.985) for the ELISA and 0.915 (95% CI: 0.863-0.968) for the skin-scraping method. By logistic regression analysis, one explanatory biological factor-result to the skin-scraping method-and four explanatory biological factors-Tyroglyphidae individual status, Trichophyton verrucosum individual status, Oestrus ovis status of the flock and presence of adjacent animals with a clinical disease neighbour to S. scabiei infection-were found for diagnostic sensitivity and specificity of the ELISA, respectively, although this depended on the OD% cut-off value used. Latent-class analysis, carried out for the ELISA at 17.8 OD% cut-off value (mean plus 3 SDs of sheep considered negative to anti-S. scabiei antibodies), showed a marked difference between the estimated diagnostic sensitivity of the ELISA (87.6%) and the skin-scraping method (62.8%), but closer diagnostic specificities (95.9% vs. 100%, respectively). These results demonstrate that the developed ELISA is valid for different applications in clinical as well as in epidemiological contexts.

Clinical evaluation and antibody responses in sheep after primary and secondary experimental challenges with the mange mite Sarcoptes scabiei var. ovis.

In this work the clinical evolution and the specific serum IgG and IgE antibody responses in sheep after primary (n=10) and secondary (n=4) experimental challenges with the mange mite Sarcoptes scabiei var. ovis were studied. The primary infection was characterized by the development of mange lesions in all sheep, a detection of live S. scabiei mites in 70% skin scrapings taken in week 10 post-challenge (PC), strongly raised and sustained specific IgG levels and a more moderate but continuous rise in specific IgE levels. Seroconversion was detected for IgG and IgE by ELISA in 90% and 60% of the sheep in week 8 PC, respectively. By Western-blotting (WB), ten IgG-reactive bands (36-120 kDa) and four IgE-reactive bands (90-180 kDa) were observed in week 8 PC. Following the secondary challenge the ewes developed a smaller area of mange lesion than that seen following primary challenge and live S. scabiei mites were not detected in skin scrapings collected in week 8 PC, suggesting that sheep had developed immunity to re-infection. Compared to primary infection, the specific IgG secondary antibody levels were transient, but in contrast there was an anamnestic IgE response, resulting in an elicitation of specific serum IgE levels in week 2 PC significantly higher than those demonstrated after primary infection. WB analysis revealed one additional IgG-reactive band (180 kDa) and no additional IgE-reactive bands. Determining the immunodiagnostic or vaccination value of the IgG-reactive antigens and IgE-reactive allergens detected requires further studies.