Mean corpuscular haemoglobin concentration as haematological marker to detect changes in red blood cells in sheep infected with Haemonchus contortus.

Haemonchus contortus is a nematode parasite that causes anaemia and affects the health of sheep. The mean corpuscular haemoglobin concentration (MCHC) is an excellent indicator to detect anaemia that could help to characterize resistant or susceptible lambs to gastrointestinal nematodes. The aim of this study was to evaluate the predictive value of MCHC in detecting changes in red blood cells and their relation to anaemia in lambs re-infected with H. contortus. An analysis of information was performed using 24 Pelibuey lambs previously infected in grazing, dewormed and experimentally re-infected with H. contortus. At the first haematological sampling (admission) the lambs were classified based on MCHC quartiles (Q). Subsequently, the lambs were housed for 56 days. Blood samples were taken every seven days to determine the haematological parameters using an impedance haematological instrument. Confidence limits were constructed with the records of the lambs that recovered their haematological parameters. Each quartile was analysed as a treatment in a repeated measures design over time. To know the optimal combination of sensitivity and specificity of MCHC to detect anaemia a curve of receiver operating characteristic (ROC) curve and the cut-off values were evaluated. In quartile 4 (Q4), lambs showed the highest faecal egg count (FEC, 764 eggs/g of faeces), mean corpuscular haemoglobin (17.0 pg) and MCHC (54.6 g/dL). This group also presented the lowest RBC values (5.8 × 106/mL), haematocrit (HCT, 18.3%), total plasma protein (5.7 g/dL), and HGB (9.7 g/dL). The optimal point of MCHC with ROC curve was 42.4 (sensitivity 88.2% and specificity 86.5%); the area under the curve was 0.91 (CI 95%, 0.86-0.96). These results are related to the haematological effects caused by H. contortus in susceptible lambs. In conclusion, the highest FEC and lower HCT in Q4 are important elements of the haematological damage caused by H. contortus and could identify susceptible lambs.

Haematological variables of Santa Ines and Ile de France suckling lambs: Influence of Haemonchus contortus infection / Variáveis hematológicas de cordeiros lactentes Santa Inês e Ile de France: influência da infecção por Haemonchus contortus

Haemonchus contortus is the major gastrointestinal parasite of sheep raised in tropical and subtropical areas worldwide. This trial aimed to evaluate the influence of H. contortus infection on the bone marrow response of Santa Ines (SI) and Ile de France (IF) suckling lambs experimentally infected with H. contortus. Fourteen SI lambs and 12 IF lambs were randomized in four groups: infected SI (n=8), non-infected SI (n=6), infected IF (n=8) and non-infected IF (n=4). Lambs of infected groups were submitted to 27 infections, conducted every two days, from 14 to 68 days of age, and each lamb received a total of 5400 H. contortus infective larvae (L3). Ten blood samples were obtained during the experimental period to be used for erythrocyte and leukocyte counts, packed cell volume and total plasma protein estimation. Additionally, it was carried out a differential leukocyte count. Lambs from control groups did not shed eggs in faeces all over the experiment, while infected Santa Ines and Ile de France lambs presented means of 2963 EPG (Eggs Per Gram of faeces) and 8175 EPG in the last sampling (P<0.05), respectively. Infected Santa Ines lambs had an increase in eosinophil release, however differences (P<0.05) on circulation number in comparison with infected Ile de France lambs were identified only in the last sampling (54 days post first infection). The mild H. cortortus infection did not produce significant changes in the blood variables of the Ile de France and Santa Ines suckling lambs.(AU)

Haemonchus contortus é o principal parasita gastrintestinal de ovinos criados em áreas tropicais e subtropicais. Este estudo objetivou avaliar a influência da infecção por H. contortus na resposta medular de cordeiros lactentes das raças Santa Inês (SI) e Ile de France (IF) infectados experimentalmente com H. contortus. Quatorze cordeiros SI e 12 cordeiros IF, foram distribuídos em quatro grupos: SI infectado (n=8), SI não infectado (n=6), IF infectado (n=8) e IF não infectado (n=4). Cordeiros dos grupos infectados foram submetidos a 27 infecções, realizadas a cada dois dias, do 14º até 68º dia de vida, com um total de 5400 larvas infectantes (L3) de H. contortus por animal. Foram realizadas 10 coletas de sangue ao longo do período experimental para determinação do volume globular, proteínas plasmáticas totais, contagem de eritrócitos e leucócitos, além do diferencial de leucócitos. Cordeiros controles não infectados não eliminaram ovos nas fezes durante todo o experimento, enquanto que os cordeiros Santa Ines e Ile de France infectados apresentaram em média 2963 ovos por grama de fezes (OPG) e 8175 OPG na última coleta (P<0,05), respectivamente. Cordeiros Santa Inês infectados tiveram um aumento na produção de eosinófilos, mas diferenças (P<0.05) no número de eosinófilos sanguíneos em comparação a cordeiros Ile de France infectados foram detectadas somente na última coleta (54 dias após a primeira infecção). A infecção leve por H. cortortus não induziu alterações significativas nas variáveis sanguíneas de cordeiros lactentes Ile de France e Santa Inês.(AU)

Anthelmintic efficacy of trichlorfon and blood parameters of young lambs infected with Haemonchus contortus.

In this study we evaluated the efficacy of trichlorfon against Haemonchus contortus, monitoring its influence on blood parameters and plasma enzymes of lambs with haemonchosis. A lamb group was orally treated with trichlorfon at 100 mg kg-1 while the other group was untreated. Split-plot design analysis was performed with the lamb groups defined as plots while the subplots were the four periods (weeks) of collection. The trichlorfon treatment promoted a significant and effective reduction of fecal egg counts after one week, with efficacies > 99%. After 21 days of treatment, detected blood parameters and serum levels of plasma enzymes were normal. Additionally, serum albumin and urea concentrations increased to normal values, which were not observed in untreated lambs. The treatment with this organophosphate, using a correct oral administration, may represent an effective therapeutic alternative for sheep infected with multi resistant strain of H. contortus.

Across and within breed differences in the relationship between packed cell volume and fecal egg count in growing meat goat and hair sheep males naturally and artificially infected with gastrointestinal nematodes.

The relationship between packed cell volume (PCV) and fecal egg count (FEC) in different breeds of meat goats and hair sheep infected with gastrointestinal nematodes, including Haemonchus contortus, was characterized. Growing males from eight commercial and two research farms (one Kiko, Spanish, Dorper, and St. Croix; three Boer; four Katahdin) in the southcentral United States were evaluated in a central performance test with ad libitum intake of a 50% concentrate pelleted diet. There were 84 Boer, 55 Kiko, and 57 Spanish goats and 52 Dorper, 129 Katahdin, and 49 St. Croix sheep. During adaptation, animals were dewormed then dosed with 10,000 infective H. contortus larvae. PCV and FEC were determined before deworming (i.e., natural infection potentially with multiple internal parasites) and 21, 28, 35, 42, and 49 days after artificial infection. Effects of species, breed, and year were analyzed with mixed effects models including day of sampling post dosing as a repeated measure and FEC and FEC × breed as covariates. Moreover, differences in correlation coefficients between PCV and logarithmic FEC (lnFEC) among species, breed, year, and day of sampling were evaluated. Breed affected (P ≤ 0.001) PCV in goats (24.8, 27.2, and 26.0% for Boer, Kiko, and Spanish, respectively; SEM = 0.42) and sheep (29.8, 26.7, and 31.0% for Dorper, Katahdin, and St. Croix, respectively; SEM = 0.28). There were effects of FEC × breed (P ≤ 0.029) on PCV for Boer, Kiko, Dorper, Katahdin, and St. Croix (-0.31, -0.33, -0.46, -0.46, and - 0.49% per 1000 eggs, respectively) but not for Spanish goats (P = 0.451). With all data, PCV and lnFEC with natural infection were highly correlated (P < 0.001) for Boer and Kiko goats and Dorper and Katahdin sheep (r = -0.59, -0.67, -0.77, and - 0.84, respectively) but not for Spanish goats or St. Croix sheep (P ≥ 0.323). Correlation coefficients for artificial infection with H. contortus were significant (P ≤ 0.002) except for Spanish goats, although values were lower (-0.40, -0.21, -0.23, -0.47, and - 0.28 for Boer, Kiko, Dorper, Katahdin, and St. Croix, respectively) compared with natural infection. In conclusion, PCV was not related to FEC in Spanish goats infected either naturally or artificially, and the nature of the relationship varied among breeds of goats and sheep. Based on the magnitude of the FEC × breed coefficient, sheep incurred a relatively greater reduction in PCV as FEC increased, and correlation coefficients indicate stronger relationships with natural than artificial infection.

Transcriptional profiling of innate immune responses in sheep PBMCs induced by Haemonchus contortus soluble extracts.

BACKGROUND: Pattern recognition receptors (PRRs) can recognize pathogen-associated molecular patterns and activate downstream signalling pathways, resulting in modulation of host immunity against pathogens. Here, we investigated whether PRR-mediated recognition is involved in host immune responses to the blood-feeding nematode Haemonchus contortus. METHODS: During blood-feeding, H. contortus secretes immune-modulating antigens into host blood. Therefore, we stimulated sheep peripheral blood mononuclear cells (PBMCs) with H. contortus soluble extract (HcAg) and performed transcriptional profiling. RESULTS: HcAg upregulated two genetically linked CLRs (CLEC2L and KLRG2), two NLRs attenuating inflammation (NLRP12 and NLRC3) and one G protein-coupled receptor with potent anti-inflammatory effects (HCAR2). Furthermore, several Th2-related transcription factors (ATF3, IRF4, BCL3 and NFATC) were also upregulated, which may confer anti-inflammatory type 2 immune responses to HcAg. CONCLUSIONS: Together, our preliminary studies provide new insights into how the host innate immune system controls type 2 immunity to H. contortus. Further work will be needed to identify H. contortus products recognized by the host innate immune system and determine the Th2 polarization ability of these putative PRR ligands.

Sensitivity and specificity of the FAMACHA© system in tropical hair sheep.

The aim of this research was to evaluate the sensitivity and specificity of the FAMACHA© (F©) system in Morada Nova ewes. The conjunctivae of 562 ewes were evaluated using the F© system. Packed cell volume (PCV) served as the gold standard for clinical F© evaluation. To calculate the sensitivity and specificity of the F© system, different criteria were adopted: animals classified as (I) F© 4 and 5 or (II) 3, 4, and 5 were considered to be anemic and animals classified as (I) F© 1, 2, and 3, or (II) 1 and 2 were considered to be non-anemic. Three standard values of PCV, namely, ≤ 19%, ≤ 18%, or ≤ 15%, were used to confirm anemia. The percentage of correct treatments was always high when the F© values 4 and 5 were used as criteria for positive tests. For all the PCV cut-off values, more animals were classified as false positives when evaluated using F© 3, 4, and 5 as criteria for a positive test and more true negative animals when evaluated using only F© 4 and 5 as criteria for a positive test. For both sets of criteria for the positive tests, few animals were classified as false negatives and true positives. Eliminating the classification of F© 3 as anemic decreased the sensitivity and increased the specificity for all the PCV cut-off values for the ewes. The F© system can be used as a reliable alternative to reduce selection pressure for anthelmintics in relation to routine non-selective blanket treatment for worm control in the Morada Nova ewes.

Serum-mediated Haemonchus contortus larval aggregation differs by larval stage and is enhanced by complement.

The objectives of this study were to measure Haemonchus contortus larval aggregation by complement/antibody complexes, determine effect of breed resistance and infection status and determine the effect of larval maturation on larval aggregation in vitro. Larval binding assays were performed on H. contortus L3, exsheathed L3 and L4 incubated with serum from either parasite naïve or H. contortus primed St. Croix (resistant) and Suffolk (susceptible) lambs. No differences in L3 aggregation were observed between serum from either breed or infection status. Exsheathed L3 (60%) and L4 (42%) aggregation by primed Suffolk serum was significantly reduced compared with L3 (80%, P<.001). Removal of either complement or antibody effectively eliminated L3 aggregation (P<.001). Combination of antibody-depleted and complement-inactivated serum restored L3 aggregation to levels consistent with unprocessed serum, supporting a role for antibody and complement in aggregation (P<.001). Use of fluorescence-labelled anti-sheep IgG antibody allowed documentation of IgG bound to serum complexes within L3 masses and was present only in larvae incubated with normal serum, and complement- and antibody-depleted serum combination. These data indicate that complement/antibody complexes inhibit larval motility through larval aggregation which may be critical in early larval clearance of H. contortus.

Experimental infection of calves with Haemonchus placei or Haemonchus contortus: Assessment of clinical, hematological and biochemical parameters and histopathological characteristics of abomasums.

The present study had the primary objective of evaluating clinical, hematological and biochemical parameters, as well as observing anatomical and histopathological characteristics of abomasums, from calves prime-infected with Haemonchus contortus or H. placei. Ten male Holstein newborns were subdivided in three groups (GI placebo; GII infected with H. contortus; GIII inoculated with H. placei). Eye mucosa staining was evaluated. Hematological and biochemical tests were performed on animals. The euthanasia of all ten experimental calves was performed on the 42nd day post-inoculation. Fragments were collected from each of all 10 abomasums for histopathological analysis. Discrete submandibular edema was diagnosed in animals from both infected groups (H. contortus or H. placei). However, there were no significant changes (P > 0.05) in the color of the ocular mucosa of calves from all three experimental groups across the entire experimental period. Hematological and biochemical changes diagnosed on animals could not be linked to infections by species of Haemonchus spp. Regarding histopathological exams, it was possible to diagnose hypertrophy, hyperplasia, binucleated cells, inflammatory infiltrate, multifocal hemorrhage and edema in abomasums from calves of both groups infected with H. placei and H. contortus. It can, thus, be concluded that not only are calves susceptible to infections by both Haemonchus species, but they can also present clinical changes and similar anatomic histopathological lesions independent of being infected by Haemonchus placei or Haemonchus contortus. These results reflect a negative effect on helminth control by mixed grazing between sheep and cattle, especially when using calves.

Influence of immunoprotection on genetic variability of cysteine proteinases from Haemonchus contortus adult worms.

The limitations associated with the use of anthelmintic drugs in the control of gastrotintestinal nematodosis, such as the emergence of anthelmintic resistance, have stimulated the study of the immunological control of many parasites. In the case of Haemonchus contortus, several vaccination trials using native and recombinant antigens have been conducted. A group of antigens with demonstrated immunoprotective value are cathepsin B - like proteolytic enzymes of the cysteine proteinase type. These enzymes, which have been observed in both excretory-secretory products and somatic extracts of H. contortus, may vary among different geographic isolates and on strains isolated from different hosts, or even from the same host, as has been demonstrated in some comparative studies of genetic variability. In the present study, we evaluated the genetic variability of the worms that fully developed their endogenous cycle in immunised sheep and goat in order to identify the alleles of most immunoprotective value. To address these objectives, groups of sheep and goats were immunised with PBS soluble fractions enriched for cysteine proteinases from adult worms of H. contortus from either a strain of H. contortus isolated from goats of Gran Canaria Island (SP) or a strain isolated from sheep of North America (NA). The results confirmed the immunoprophylactic value of this type of enzyme against haemonchosis in both sheep and goats in association with increased levels of specific IgG. The genetic analysis demonstrated that the immunisation had a genetic selection on proteinase-encoding genes. In all the immunised animals, allelic frequencies were statistically different from those observed in non-immunised control animals in the four analysed genes. The reduction in the allelic frequencies suggests that parasites expressing these proteases are selectively targeted by the vaccine, and hence they should be considered in any subunit vaccine approach to control haemonchosis in small ruminants.

Anthelmintic effect of carob pods and sainfoin hay when fed to lambs after experimental trickle infections with Haemonchus contortus and Trichostrongylus colubriformis.

The aim of the study was to compare the in vivo anthelmintic activity of sainfoin hay (Onobrychis viciifolia) and carob pod meal (Ceratonia siliqua) against gastrointestinal nematodes. Seven days before infection, 64 naive lambs were assigned to four different groups: Group S received sainfoin hay and group CAR was fed with carob pods. The remaining lambs received lucerne hay (Medicago sativa) and were assigned to positive (non-treated, NT) and negative (treated, T) control groups (treatment with albendazole). On day 0, lambs were artificially trickle infected for 6 weeks, with a mixture of infective larvae of Haemonchus contortus and Trichostrongylus colubriformis. Parasitological and pathophysiological parameters were measured repeatedly during the 2-month study. Compared to the NT group, decreases in egg excretion were observed in the CAR and S groups with significant differences only found for sainfoin (p < 0.05). At necropsy, group S showed decreases in the total worm numbers of both nematode species with significant differences for H. contortus. In contrast, no differences were noticed for the CAR group. Compared to the NT group, lower values for fecundity of female H. contortus were found in the S and CAR groups, however differences were non-significant. No differences in body weight gains were found between groups. Consistent results were found showing significantly higher packed cell volume (PCV) values in the T and S groups compared to NT and CAR groups. Overall, these results confirm a positive effect associated with the feeding of lambs with tanniniferous resources on host resilience (PCV values) and against gastrointestinal parasitic nematodes by affecting some biological traits of worm populations (e.g. eggs per gram of faeces and worm numbers). However, the anthelmintic effects differed between the two tannin-containing resources, which might be associated with the quantity and/or quality of secondary metabolites (condensed tannins and/or other polyphenols).

Relationship between oxidative stress and pathological findings in abomasum of infected lambs by Haemonchus contortus.

The aim of this study was to evaluate the nitric oxide (NO) levels, and oxidative and antioxidant markers of lambs experimentally and naturally infected by Haemonchus contortus, and its relation to lesions in the abomasum. For experimental study, a total of 14 healthy lambs were divided into two groups with seven animals each. Group A represented the uninfected animals (control), and Group B was formed by infected animals with 15,000 larvae of H. contortus. Blood was collected on days 15, 45, and 75 post-infection (PI) to obtain serum for biochemical analysis: lactate dehydrogenase (LDH), nitrite/nitrate (NOx), advanced oxidation protein products (AOPP), and ferric reducing ability of plasma (FRAP). Parasitological stool examination (eggs per gram of feces--EPG) was performed on days 15, 45, and 75 PI to verify the evolution of the infection. On day 15 PI EPG was negative, but on days 45 and 75 PI the EPG was positive for animals from Group B. In the three periods evaluated it was observed an increase of LDH levels in serum of lambs infected with gastrointestinal nematodes, but on the other hand NOx levels were reduced on the same periods in infected animals. The AOPP and FRAP levels did not differ between groups on days 15 and 45 PI, but increased significantly on day 75 PI in infected lambs. The same variables were studied in 10 lambs naturally infected with helminths, where more than 97% corresponded to H. contortus (hematocrit and EPG values were 18.8 ± 2.5% and 7120 ± 2940, respectively). Similar to the experimental study, the levels of NOx reduced, and the levels of LDH, FRAP, and AOPP increased in serum of this animal associated inflammatory infiltrate in the mucosa of the abomasum. Therefore, during the infection by H. contortus it was observed alterations in oxidative markers, indicators of cell lesion confirmed by histological examination of the abomasum, and consequently there were changes in antioxidant levels, with the purpose of cell protection. We also conclude that helminth infection interferes with the nitric oxide metabolism.

Experimental infection by Haemonchus contortus in lambs: influence of disease on purine levels in serum.

The aim of this study was to evaluate the purine levels of lambs experimentally infected with Haemonchus contortus. A total of 12 healthy lambs were divided into two groups, composed of 6 animals each: Group A represented the healthy animals (uninfected), while in Group B the animals were infected with 15 000 larvae of H. contortus. Blood was drawn on days 15, 45 and 75 post-infection (PI) in order to perform the purine analysis (ATP, ADP, AMP, adenosine, inosine, hypoxanthine, xanthine and uric acid) by high pressure liquid chromatography (HPLC) in serum. On day 15 PI a significant (P<0·05) increase in the levels of ATP and inosine was observed in the infected animals, unlike the levels of ADP, adenosine, xanthine and uric acid which were reduced. On day 45 PI a significant (P<0·05) increase in the ATP and xanthine levels in infected animals was observed, contrasting with reduced levels of ADP and uric acid. Finally, on day 75 PI an increase occurred in the levels of ATP, adenosine and hypoxanthine in infected lambs, concomitant with a reduction in the levels of ADP and uric acid (P<0·05). These changes in purine levels may influence the inflammatory process and the pathological events.

Influence of experimental infection by Haemonchus contortus on acetylcholinesterase activity in lymphocytes of lambs.

The aim of this study was to evaluate the acetylcholinesterase (AChE) activity in lymphocytes of lambs experimentally infected by Haemonchus contortus. A total of 14 healthy lambs were used, divided into two groups of seven animals each. Group A (negative control) represented the uninfected animals, and Group B (positive control) was formed by animals infected with 15,000 larvae of H. contortus. Blood was drawn on the days 15, 45 and 75 post-infection (PI) in order to perform the white blood cells (WBC) count, as well as the evaluation of AChE activity in lymphocytes. Parasitological stool exam (eggs per gram of feces - EPG) was performed on the same days to follow up the evolution of the infection. On day 15 PI it was verified negative EPG; however, on days 45 and 75 PI it was observed positive EPG only in the animals of group B. In the three evaluated periods was observed a lower number of leukocytes, associated with decreased lymphocytes and neutrophils in lambs infected by this gastrointestinal nematodes. Lambs infected with H. contortus showed significant (P<0.01) lower AChE activity in lymphocytes compared uninfected. Statistically, there was a positive correlation (P<0.05) between AChE activity in lymphocytes and number of lymphocytes (r=0.69). The lymphocytes are cells with direct participation in the cholinergic system; therefore, based on these results, it can be concluded that the experimental infection with H. contortus influences the number of lymphocytes, and consequently the AChE activity in these cells.

Effect of experimental infection with Haemonchus contortus on parasitological and local cellular responses in resistant and susceptible young Creole goats.

This study was carried out to evaluate the relationships of cellular changes in the abomasal mucosa and parasitological parameters, by comparing resistant and susceptible young Creole goats (kids) after experimental infection with Haemonchus contortus. The kids were infected over 2 periods (challenges 1 and 2) of 7 and 6 weeks, respectively. Fecal egg count (FEC), blood eosinophilia, packed cell volume (PCV), and body weight were weekly monitored. At the end of both challenges a subgroup of kids was slaughtered for nematode burden measurements and analysis of inflammatory cell infiltration in the abomasal mucosa. The average daily gain was higher in resistant kids after both challenges. Blood eosinophilia and FEC were higher in susceptible kids after both challenges. The number of immature worms and the means of female length were lower after challenge 2 whatever the genetic status. No differences were observed in the eosinophil and mononuclear cell infiltration between challenges 1 and 2 and resistant and susceptible kids. Globule leukocyte infiltration was found higher after the challenge 1 in resistant kids. This effect of the genetic status on globule leukocytes counts but not on the other inflammatory cell highlights the need for further study on the functional activity of these cell populations.

E-ADA activity in serum of lambs experimentally infected with Haemonchus contortus.

The aim of this study was to evaluate adenosine deaminase (E-ADA) activity in sera of lambs experimentally infected with Haemonchus contortus. We used 12 lambs divided into 2 groups; Group A had 5 healthy, non-infected animals (control) and Group B had 7 healthy animals infected with H. contortus . Lambs were infected orally with 500 larvae (L3) per animal every 2 days, for a period of 20 days, and later the infection was confirmed by examination of feces (eggs per gram [EPG] via fecal egg count). Blood collection was performed at days 0, 20, 40, 60, and 80 post-infection (PI) for analysis of E-ADA activity. Animals in Group A showed negative EPG throughout the experiment unlike those from Group B that had elevated EPG counts. E-ADA activity was reduced in the serum of animals infected with H. contortus when compared to non-infected controls at days 20, 40, 60, and 80 PI. Therefore, it is concluded that infection with H. contortus influences the E-ADA activity in lambs.

Effects of single or trickle Haemonchus contortus experimental infection on digestibility and host responses of naïve Creole kids reared indoor.

The objective of this study was to compare the effects of the type of Haemonchus contortus experimental infection (trickle infection, TI versus single infection, SI) on feed intake, nutrients digestibility, parasitological and haematological measures, and plasma leptin in Creole kids. The animals were infected over 2 periods (challenge 1 and challenge 2) of 6 weeks each, corresponding respectively to the primary and the secondary infection. Periods prior infection (1 week each) were considered as controls. The primary infection was realized with 35 Creole kids (18.40±3.76 kg BW) housed in individual boxes and fed a hay-based diet. The secondary infection continued with 29 kids (21.90±3.40 kg BW) from the initial 35. A total of 6 kids and 8 kids were slaughtered for measuring nematode burden at the end of the primary and the secondary infection, respectively. Measurements of nutrients digestibility were made at 0, 3 and 5 weeks post-infection for both challenges. Faecal egg count (FEC), blood eosinophilia and packed cell volume (PCV) were monitored weekly. Feed intake (dry matter intake, DMI) and nutrients digestibility were negatively affected by H. contortus infection only during the primary infection. Plasma leptin changed significantly over time (P=0.0002) but was not affected by the infection type. Effect of infection type was observed only on crude protein digestibility during the primary infection, which was lower in the TI group (P<0.01). The overall level of blood eosinophilia was significantly higher in the TI group (P<0.0001) during both challenges. The overall FEC mean was significantly higher in the SI compared with the TI groups, during both challenges (P<0.02). These results were related to the mean female length significantly higher in the SI group compared with the TI group during challenge 1 (P=0.004), and the number of adult nematode significantly lower in the TI group compared with the SI group during the challenge 2 (P=0.05). The results showed that the response of Creole kids to H. contortus experimental infection was in part dependent on the type of experimental infection. Our data suggest that plasma leptin would not be involved in the response of Creole kids against H. contortus infection, as no relationship between its plasma level and the transient reduction in voluntary feed intake observed in both groups during the primary infection was observed.

Immunoprotective effect of cysteine proteinase fractions from two Haemonchus contortus strains adapted to sheep and goats.

A preliminary analysis of the significance of genetic diversity in cysteine proteinase genes has been performed simultaneously in sheep and goats, with regard to the immunological control using these enzymes against haemonchosis. For this purpose, we have studied the cross-immunoprotective effect of cysteine protease-enriched protein fractions (CPFs) in adult worms of two Haemonchus contortus strains from North America and Spain that are adapted to sheep and goats, respectively. Previous genetic analysis of cysteine proteinase genes in both strains has shown that some of loci are polymorphic and these differences are translated into changes in the amino acid sequences. However, our results show that CPFs from H. contortus adult worms have a protective effect against the parasite in both sheep and goats. These results are similar regardless of whether they were obtained from sheep or goat-adapted H. contortus strains, which could be very important in case H. contortus CPFs were commercially used in different countries, as vaccines to prevent the negative effects of this parasite. Interestingly, this experimental inoculation of both species with a heterologous strain of H. contortus contributes to the idea shown in previous studies about how difficult is the interpretation and the comparison of vaccination where strains not adapted to a specific host are used. Therefore, the challenger of using heterologous strains could provide similar results to those observed in immunised animals. This study suggests the possibility of exploring the mechanisms involved in natural protection against non-adapted strains, in order to develop strategies to control haemonchosis.

Influence of protein supplementation during late pregnancy and lactation on the resistance of Santa Ines and Ile de France ewes to Haemonchus contortus.

This experiment aimed to evaluate the effect of periparturient metabolizable protein (MP) nutrition on resistance to Haemonchus contortus in single rearing Ile de France and Santa Ines ewes. The restriction-fed iso-energetic diet was calculated to provide either 0.8 (low MP diet) or 1.3 (high MP diet) times MP, from three weeks before parturition until eight weeks into lactation. The ewes were experimentally infected with 1000 H. contortus infective larvae (L3) three times a week (Mondays, Wednesdays and Fridays), starting five weeks before the predicted date for parturition until a total of 15,000 L3 had been administered. While both breeds showed elevated fecal egg counts (FEC), these values were significantly lower for Santa Ines ewes than Ile de France ewes, but were independent of level of MP feeding. The latter also did not affect lamb weight gain and ewe body weight variation in each breed. Packed cell volume and total plasma protein for Santa Ines in all periods were significantly higher than those for Ile de France ewes (P<0.01) but were not affected by nutrition. In contrast, levels of serum IgG and IgA antibodies against somatic H. contortus infective larvae and adult antigens were similar between breeds but higher in animals that received high MP diets (P<0.05). The reduced body score of ewes at the beginning of the experiment probably influenced their high susceptibility to incoming larvae. Since, unexpectedly, MP scarcity was not achieved in this experiment, our data support the view that Santa Ines ewes are more resistant to H. contortus than Ile de France ewes.

Comparative drug systemic exposure and clinical efficacy against resistant nematodes in lambs treated with different albendazole formulations.

A pharmaco-parasitological assessment of four different albendazole (ABZ) formulations was carried out in lambs infected with multiple resistant gastrointestinal (GI) nematodes. The comparative drug systemic exposure profiles (ABZ sulphoxide plasma concentrations) and anthelmintic efficacies (clinical endpoint measured through the faecal nematode eggs reduction counts) were determined for a reference formulation (RF) and three different test (T1, T2, T3) generic ABZ preparations. Fifty (50) Corriedale lambs naturally infected with multiple resistant GI nematodes were allocated into five experimental groups (n = 10). Animals in each group received treatment with either the RF, one of the test ABZ formulations (5 mg/kg by the intraruminal route) or were kept as untreated control. Blood samples were collected over 48 h post-treatment. ABZ parent drug was not recovered in the bloodstream. The ABZ sulphoxide (ABZSO) and sulphone (ABZSO(2) ) metabolites were measured in plasma by ultraviolet high-performance liquid chromatography over 36-48 h post-treatment. A faecal nematode egg count reduction test (FECRT) was performed at day 10th post-treatment to lambs from all treated and untreated groups, which indicated the predominance of nematodes with high level of resistance to ABZ. Both ABZSO C(max) and AUC(0-LOQ) values obtained for the RF (pioneer product) were significantly higher (P < 0.05) than those obtained for the T1 and T3 preparations. Based on the currently available bioequivalence criteria, the test (generic) ABZ formulations under evaluation could not be considered equivalent to the RF regarding the rate (C(max) ) and extent (AUC(0-LOD) ) of drug absorption (indirectly estimated through the ABZSO metabolite). A large variation in nematode egg counts did not permit to obtain statistically significant differences among formulations. However, a favourable trend in the efficacy against the most resistant nematodes was observed for the formulations with the highest ABZSO systemic exposure.

Application of ROC curve analysis to FAMACHA© evaluation of haemonchosis on two sheep farms in South Africa.

Test sensitivity and specificity for the FAMACHA(©) clinical test for anaemia due to haemonchosis have previously been shown to be adequate in differentiating between heavily/less heavily infected sheep, but these properties give no objective guidance for setting the optimum threshold at which anthelmintic treatment should occur. The aim of this work was to use Receiver Operating Characteristic (ROC) curves to evaluate the diagnostic accuracy of FAMACHA(©) testing by estimating the area under the ROC curve, and to use two-graph ROC curves to decrease subjectivity in selecting treatment thresholds on two farms with contrasting management. Test diagnostic accuracy, and thus discriminating power as determined by the area under the ROC curves, ranged from "moderate to good" on the first farm, and from "moderate to high" on the second farm for haematocrit (the Gold Standard for the test) cut-offs of ≤ 22% and ≤ 19% on both farms respectively. Accuracy of classification between haematocrit cut-offs was not significantly different within farms, but did differ significantly between farms, with test accuracy being highest on the second farm at both haematocrit cut-offs (p<0.05). The results also showed the suitability of the two-graph ROC curve approach for discriminating not only between different levels of accuracy of evaluators, but also to give an indication of the so-called ROC cut point (i.e. the desired threshold level) at which animals should be treated for a given level of risk of loss. The approach appears to have the potential not only to validate the diagnostic accuracy of the test across the complete testing range (i.e. all FAMACHA(©) categories from 1 to 5), but also to compensate for such inaccuracy by allowing objective adjustment of the threshold treatment level according to the output of the two-graph ROC method.