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.

Evaluation of official eradication-campaigns data for investigating small-ruminant brucellosis in the province of León, Spain.

Brucellosis official eradication-campaigns data were used to study the epidemiology of the small-ruminant brucellosis infection in the province of León (northwest Spain). Data from three consecutive campaigns (1997-1999) were retrieved from flock sheets from controlled flocks and from other official statistics. In general, flock information was scarce, with a small percentage of contradictory data and other deficiencies that might have affected the campaign results. Nevertheless, unconditional multivariable logistic regression showed four risk factors for brucellosis infection in 1999: larger flock size, higher percentage of flocks in the town that are seropositive and having been brucellosis positive in 1997 and 1998. Despite of the scarcity of data available from official records, they were useful to explain, in part, the extent of the SRB in the province of León in 1999.

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.

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.

Obtaining hyperimmune anti-Cryptosporidium parvum ovine colostrum. A study of the humoral immune response in immunized sheep.

Three ewes were immunized five times over a 2-month period prior to giving birth by intramuscular injection, oral administration and intramammary infusion of antigen and viable or freeze-dried Cryptosporidium parvum oocyst solution emulsified with Freund's complete and incomplete adjuvant. Two animals served as controls and another two as adjuvant controls. Serum was collected at first immunization and thereafter every 2 to 4 weeks. Colostrum and milk were collected as well. All samples were assayed for C. parvum-specific antibodies using an enzyme-linked immunosorbent assay methodology, and Western blotting was used to recognize the C. parvum antigens. Hyperimmunization resulted in a progressive and significant increase in specific anti-C. parvum serum IgG, IgA and IgM titres, with the highest values noted at the point of lambing. Titres decreased slightly in milk, although they were in all cases higher than those in the control animals. Moreover, some 30 bands of C. parvum were recognized.

Anthelmintic resistance in trichostrongylid nematodes of sheep farms in Northwest Spain.

A survey to determine the prevalence of anthelmintic resistance in nematode parasites of sheep against the three main families of anthelmintics (benzimidazoles, macrocyclic lactones and imidazothiazoles) was carried out from January 1999 to December 2003 involving 85 flocks in Northwest (NW) Spain. In the study on prevalence of resistance to benzimidazoles, faecal egg count reduction test (FECRT) showed there was resistance in 8 (12.7%) flocks, 1 (1.6%) was suspected, and 54 (85.7%) were susceptible. The results indicated that 9 flocks (34.61%) showed resistance, 1 (3.85%) showed suspected resistance, and 16 (61.54%) were susceptible to the imidazothiazoles. Against macrocyclic lactones, resistance was observed in 8 flocks (15.69%), 4 (7.84%) showed suspected resistance and 39 (76.47%) were susceptible. None of the flocks used in the study showed resistance to the three families of anthelmintics. Nevertheless, six were recorded as resistant or suspected of being resistant to two of these families of anthelmintics. Egg hatch assay (EHA) and FECRT were carried out jointly on 61 flocks, although EHA was done on a total of 83 farms. The results showed that 15 (18.07%) of the 83 flocks were resistant, with egg death 50 over 0.1 microg/ml thiabendazole, and 68 (81.93%) were susceptible to benzimidazoles. When the results between FECRT and EHA were compared, both techniques showed good correlation in field studies. Faecal cultures performed pre- and post-treatment indicated that Teladorsagia and Trichostrongylus were the main genera.

The larval feeding inhibition assay for the diagnosis of nematode anthelmintic resistance.

A larval feeding assay for detection of nematode anthelmintic resistance to macrocyclic lactones and imidazothiazoles is described. The estimated concentration of anthelmintic required to inhibit larval feeding in 50% of L1's (IC50) that were resistant to either macrocyclic lactones or imidazothiazoles were significantly higher (P < or = 0.05) than those of susceptible isolates. Some variations in IC50 values were observed during the patent period of infection in all strains, although the pattern of the IC50 followed the same course. IC50 values varied in larvae developing from eggs shed throughout the patent period, with low values in the earliest larvae followed by higher values as the infection progressed, before decreasing at 70-90 days post-infection, although the low values of the first part of the patent period were not reached. However, the IC50 differences between all resistant and susceptible strains were significant throughout the whole patent period for ivermectin and levamisole. These results suggest that this technique may provide an alternative in vitro to detect anthelmintic resistance.