Immune responses of sheep to microdissected parts of Haemonchus contortus.

Adult specimens of Haemonchus contortus were microdissected into four body fragments: oesophagus, cuticle of oesophagus, gut and cuticle with adjacent muscle layer. The antigenicity of these different body fragments was analysed in comparison to total (whole) worm extracts with immunoblotting and ELISA using sera of H. contortus-infected sheep. In particular, oesophagus-derived antigens appeared to be specifically recognized and may prove valuable in diagnostic assays.

Serum opsonic activity and neutrophil phagocytic capacity of newborn lambs before and 24-36 h after colostrum uptake.

Neutrophil (PMN) counts, immune complex (IC) uptake by PMN, and serum opsonising activity for promoting yeast uptake were used to evaluate infection clearing capacity in 16 lambs prior to colostrum feeding (two lambs fed bovine colostrum, 14 suckled lambs) and at 2 days of age. At 2 days of age lambs had more circulating PMN than they had prior to colostrum uptake (P less than 0.01). Colostrum feeding caused a significant increase in the percent of lamb PMN phagocytosing IC, although at Day 2 the percent phagocytosis was significantly lower (32.2%) than for adult controls (90%). Yeast opsonophagocytosis was greater when 24-36 h post-feeding serum was the source of opsonin than when pre-feeding serum was used (P less than 0.001). When adult serum was the opsonin, yeast opsonophagocytosis was approximately twice the phagocytosis mediated by 24-36 h post-feeding serum. The peripheral neutrocytosis and the enhancement of opsonophagocytosis generated by absorption of either ovine or bovine colostrum did not differ. The results of this study suggest that the parameters evaluated may be used for indicating the presence (or absence) of passively acquired protective immunity.

Isotype-specific serum antibody responses of sheep to Haemonchus contortus antigens.

In total, 19 8-month-old Texel sheep were used to study the isotype-specific serum antibody responses against infective larvae and adult worms of Haemonchus contortus. Group Group 1 sheep (n = 7) were infected with 20,000 L3 larvae (Week 0), treated with ivermectin 6 weeks post-infection and subsequently challenged at Week 10 of the experiment. This challenge consisted of a trickle infection of 10,000 L3 larvae per week for 5 weeks. Group 2 sheep (n = 7) received a single infection at Week 10 of the experiment, and Group 3 (n = 5) served as a non-infected control group throughout the entire experiment. Individual blood and faeces samples were collected at weekly intervals. The immune responses were monitored by ELISA and Western blotting. The secondary immune response coincided with a significant reduction of the Haemonchus egg output and reduction of worm counts. Both primary and challenge infections induced humoral immune responses, and ELISA revealed that the most dominant serum antibody responses belong to the IgG1 isotype and to a lesser extent to IgG2. IgM and IgA responses were less dominant. Western blotting experiments demonstrated that many antigens were commonly recognized by antibodies from both primary and challenge infected animals. However, sera of immune animals specifically reacted with low molecular weight proteins. In particular, a 24 kDa antigen present in adult worms appeared to be specifically recognized.

Humoral responses in rabbits immunized with two fractions of Haemonchus contortus: the antigen specificity of such antibodies.

Humoral responses were examined in rabbits immunized with either 28-40 kDa (Fraction 1) or a 19-24 kDa (Fraction 2) antigenic fraction from soluble antigens (Sol L3 Ag) from infective larvae (L3) of Haemonchus contortus. These fractions were eluted from electrophoretically separated Sol L3 Ag. Immunoblots revealed antibodies to Fraction 1 (fr. 1) or Fraction 2 (fr. 2) polypeptides as well as to several other molecular weight polypeptides of the Sol L3 Ag. The latter antibodies were shown by absorption studies not to be Sol L3 Ag cross-reactive anti-bacterial rabbit antibodies. When Sol L3 Ag was affinity-purified using monoclonal antibody to phosphorylcholine (PC) and the resulting fractions were further analysed by immunoblotting using rabbit anti fr. 1 or anti fr. 2 antiserum, the PC antigen was found to be shared between fr. 1 and other polypeptides of Sol L3 Ag. Using the rabbit antibody fractions eluted from nitrocellulose membranes containing fr. 1 or 2 polypeptides, it was found that these fractions contained antibody that bound mainly to fr. 1 and only to fr. 2 polypeptides of Sol L3 Ag. It is concluded that, from the present immune rabbit sera, antibodies specific for either fr. 1 or fr. 2 may be isolated and then used to purify small amounts of the corresponding antigens.

The epidemiology of gastrointestinal nematode infections in communal cattle and commercial beef cattle on the highveld of Zimbabwe.

An epidemiological study of gastrointestinal nematode infections of cattle was conducted on the highveld of Zimbabwe from June 1993 to May 1995. The study was carried out in two communal areas, two conventional beef farms and two commercial beef farms with irrigated pastures. On all farms/areas, faecal egg counts were low (< 500 eggs per g faeces) during the dry season. During the rainy season faecal egg counts were highest in communal areas and lowest in conventional beef farms. Those of irrigated farms had intermediate values. During the dry season pasture larval counts were low in irrigated pastures and conventional beef farms and virtually zero in communal areas. They increased and peaked during the rainy season, coinciding with the egg count peaks. Worm burdens of necropsied cattle indicated that 100% of the animals were infected with nematodes. The important species were Cooperia pectinata, C. punctata, Haemonchus placei, Trichostrongylus axei and Oesophagostomum radiatum in all farms/areas and Ostertagia ostertagi in a beef farm with irrigated pastures. Haemonchus survived the dry season as inhibited early fourth stage larvae whereas Cooperia and Trichostrongylus survived as adults.

Bovine humoral immune responses to Haemonchus placei excretory secretory antigens.

The immune response of calves against excretory secretory (ES) products of adult Haemonchus placei was studied. The ES products of adult H. placei comprise at least ten polypeptides with molecular weights ranging from 10 to over 100 kDa. A primary infection of calves with H. placei did not result in a marked elevation of IgG serum antibodies reactive to ES antigens as demonstrated by ELISA. Immunoblotting experiments revealed that sera of primary infected calves recognised a 50 kDa antigen in most cases. An increase in the level of serum IgG antibodies was observed after trickle infections. The secondary immune response coincided with the recognition of a group of three ES antigens of around 50-55 kDa and one protein with a molecular weight of 24 kDa.

Control of Sarcoptes scabiei var. suis with ivermectin: influence on scratching behaviour of fattening pigs and occurrence of dermatitis at slaughter.

The behaviour of fattening pigs, the occurrence of erythematous papular dermatitis in pigs at slaughter and the effects of treatment for mange with ivermectin at the start of the fattening period were evaluated at ten farms. At each farm, trial pigs were randomly allotted to a control or a treated group. At the start of the trial, the control group was injected intramuscularly with 5 mg kg-1 levamisole, and the treated group was injected subcutaneously with 300 micrograms kg-1 ivermectin. Skin scrapings, taken from each pig before medication at the start of the trial, and at slaughter, were examined for presence of sarcoptic mites. Pig behaviour was monitored at 2 week intervals from Week 5 to Week 13 of the trial. Carcasses of trial pigs were inspected for dermatitis at slaughter. Low to moderate Sarcoptes scabiei var. suis infestations were demonstrated at the start of the trial on five farms (S+ farms). At slaughter, the mean percentage of Sarcoptes-positive pigs per pen on S+ farms was 34.8% (range 11-60%) for the control group as compared with 0.5% (range 0-2.7%) for the ivermectin-treated group (P < 0.01). No S. scabiei was recovered from any pig at any time from the five other farms (mange-free (S-) farms). The low initial levels of mange in the controls at S+ farms resulted in a consistently high scratching index. Ivermectin treatment resulted in a significantly (P < 0.01) lower prevalence of scratching, comparable with the prevalence observed at S- farms. High prevalences of generalized dermatitis at slaughter were observed in control pigs from all S+ farms. Ivermectin treatment resulted in much lower prevalences, reflected in a significantly (P < 0.01) lower grand mean dermatitis index per pen for this group as compared with the controls. The majority of pigs from both treatment groups at all S- farms were free of papular lesions. These results indicate that low levels of sarcoptic mange at the start of fattening, left untreated, will result in a high prevalence of scratching during the fattening period and high prevalences of Sarcoptes and dermatitis at slaughter. Treatment with ivermectin at the start of fattening results in behaviour and dermatitis prevalences similar to those observed in mange-free herds.

Is there a relationship between haemoglobin genotype and the innate resistance to experimental Haemonchus contortus infection in Merino lambs?

Responses to a single or repeated infection with 7000 infective larvae of Haemonchus contortus were studied in an experiment using a total of 106 3-month-old lambs with AA, AB or BB haemoglobin (Hb) genotypes. Results were assessed by faecal egg counts, adult worm counts, haematocrit values, haemoglobin concentrations, total serum protein and serum antibody IgG1 and IgA ELISA titres. None of these parameters showed a strong relationship to the Hb type. The prevalence of low responder (greater than 500 worms) and of high responder (less than 50 worms) animals in groups AA, AB and BB Hb types was 3.8 and 34.6, 20.6 and 35.2, 28.1 and 43.7%, respectively, suggesting that the responsiveness to nematode infection is under the control of gene(s) not closely linked with those determining the Hb genotype. Worm counts of a primary infection are more subject to variation than those of a secondary infection. There is a strong relationship between adult worm counts and faecal egg counts taken close to the time of slaughter. In living animals low and high responder discrimination can be based on individual faecal egg counts around 50 days after a secondary infection. Haematocrit values proved to be of little value in the low and high responder selection. In this regard neither Hb concentration nor total serum protein values are of practical significance. In 3-month-old lambs primary infection induced partial immunity which could prevent the establishment of a part of the secondary infection, irrespective of the presence or absence of the primary worm population. The development of immunity was not associated with an increase of serum IgG1 and IgA antibody levels. Specific antibody production was not influenced by Hb types. Mean antibody levels of low responder lambs showed no difference from those of high responders. Thus, serum IgG1 and IgA levels are of no predictive value in identifying lambs which are genetically resistant to Haemonchus infection.

Recovery of different stages of Dictyocaulus viviparus from cattle lungs by a combination of a perfusion and a Baermann technique.

The lungs of 71 calves used in two grazing studies were examined for lungworms with a perfusion method combined with a modified Baermann method. The numbers of worms recovered from the lungs were 11,630 in total, 7495 adults, 872 juvenile fifth stage larvae (L5) and 3263 inhibited L5. The percentages found after perfusion were 78.9 per cent in total, 91.8 per cent adults, 78.6 per cent juvenile L5 and 49.6 per cent inhibited L5. The perfusion method seemed adequate and rapid for recovering the adult and juvenile stages but not the inhibited stage. For estimating the numbers of inhibited larvae a combination of perfusion with the Baermann technique is necessary.

Serum antibody responses of Texel sheep experimentally infected with Haemonchus contortus.

The primary and secondary serum antibody responses of Texel sheep to infective larvae (L3) and adult worms of Haemonchus contortus were studied. Ten-month-old sheep were infected with 20,000 H contortus L3, treated with ivermectin seven weeks later and, after four weeks, reinfected with 10,000 L3 once a week for six weeks. Faecal egg counts were significantly lower during the secondary infection than during the primary infection, but both infections induced antibody responses, as demonstrated by an enzyme-linked immunosorbent assay (ELISA). The primary antibody response developed rather slowly, but the secondary response developed more rapidly and the IgA responses against L3 antigens and the IgG1 and IgG2 responses against adult antigens were twice those observed during the primary infection. These accelerated and enhanced responses after the reinfection suggest an immunological memory for H contortus antigens.

Prevalence of benzimidazole resistance of nematodes in sheep in The Netherlands.

A survey was carried out in The Netherlands to determine the prevalence of benzimidazole resistance in sheep nematodes. It was shown that 47 per cent of the 59 investigated flocks harboured trichostrongylid populations with resistance to benzimidazoles, as demonstrated by an LD50 value of 0.12 micrograms ml-1 thiabendazole or higher in an in vitro egg hatch assay. On these farms Haemonchus contortus was found to be the dominant species. In two flocks resistant Ostertagia and, or, Trichostrongylus species populations were found. Farms with resistance were revisited to determine the anthelmintic efficacy in vivo by means of treatment and egg count. In only four of the 19 flocks which showed resistance in vitro was an efficacy of less than 95 per cent in vivo achieved. This means that there is a discrepancy between the in vivo and in vitro results. Resistance was found more frequently on pure sheep farms than on mixed farms. It is suggested that mixed farms offer more refuge for susceptible H contortus populations.

A Hypoderma diana (Diptera: Hypodermatidae) infection in a horse.

An infection with second-stage larvae of the warble-fly H. diana in a horse is described. The second-stage larvae were incapable of developing into the third stage, because horses are unsuitable hosts and because the infected horse was treated with an insecticide. Since the horse was used for dragging trees in the forests, the infection was likely contracted via contact with H. diana, a normal parasite of roe deer in the Netherlands.

[The nematode Haemonchus contortus: antigenic characterization and immune response in rabbits and sheep]. / De nematode Haemonchus contortus: antigeenkarakter en immuunrespons bij konijnen en schapen.

In the first part of the study, various tissues of L3 larvae and adult Haemonchus contortus were examined by indirect fluorescence to investigate their antigenicity. L3 larvae were studied in immunoblots as well. The following tissues contained antigens: the sheath and cuticle of L3 larvae; and the cuticle, muscles, epithelium and glandular tissue of the oesophagus and sheath of the spicules of adult nematodes. On the cuticle of exsheathed L3 larvae, we detected by light-microscope slightly raised patches: places where the cuticle differs from the surrounding cuticular morphology. We suggest that the patches are comparable to those found on exsheathed L3 larvae of the horse. Whether the patches produce excretory or secretory antigens or both is unknown, however. Somatic soluble extracts of L3 and adult worms were found to be complex mixtures of proteins by polyacrylamide gel electrophoresis. Results in immunoblots of L3 larvae suggested that the region between the 42 kDa protein and the 18 kDa protein may contain antigens specific for H. contortus. We injected rabbits with the 42-25 kDa fraction of the L3 larvae. The rabbit sera were probed in immunoblots with the total somatic soluble L3 extract for the presence of antibodies. Antibodies reacted with antigens in the 42-25 kDa fraction, however, cross-reactions occurred with antigens in the L3 fractions that were not injected into the rabbits. We found that antibodies against phosphorylcholine were induced and suggest that these cause some of the cross-reactions. In the second part of the study sheep were infected orally with H. contortus and their lymphocyte responses towards L3 antigen and mitogen were measured in a lymphocyte stimulation test. Lymphocyte responses to L3 antigen are expressed in a stimulation index. Two groups of worm-free raised sheep were inoculated (sheep older than 1 year and lambs that were 4 to 6 months old) as well as a group of hyperimmunized sheep. Some sheep were inoculated only once; others were inoculated and then challenged. Lymphocytes of sheep that were hyperimmunized replicated rapidly in the test resulting in significantly higher stimulation indices than those recorded for the lymphocytes of all other sheep. After inoculation the enhanced response further increased. We explain these lymphocyte responses as a result from the immunological memory induced during the hyperimmunization. Lymphocytes of sheep over 1 year old only respond in the test when sheep are infected.(ABSTRACT TRUNCATED AT 400 WORDS)

[A case of demodicosis on a Dutch dairy farm]. / Een geval van demodicose bij Nederlands melkvee.

Demodex bovis is a burrowing mite that lives in the hair follicles of cattle, resulting in nodules, granulomatous inflammation, and the formation of scar tissue. Clinical and histopathological changes of a recently diagnosed case of demodicosis on a Dutch dairy farm are presented before and after treatment. Treatment results were disappointing. The role of demodicosis in Dutch dairy farming is discussed.

[Pros and cons of anthelmintic treatment of lambing ewes]. / De voor- en nadelen van een wormbehandeling van ooien bij de partus.

The advantages, prevention of gastro-intestinal worm infections in lambs, the improvement in the condition of the ewes during lactation, and the disadvantages, the costs, a possible interference with the build-up of immunity and the risks of the occurrence of anthelmintic resistance are discussed. It is concluded that the advantages more than counterbalance the disadvantages. Providing that the dosage of anthelmintics is correct, that is to say that an overdosage is preferable to too low a dosage, and safe pastures are used, post-lambing anthelmintic treatment of ewes is an effective procedure in preventing gastro-intestinal infections in lambs. Moreover, treatment will eventually improve the condition of the ewes so that, for instance, as the result of a slightly higher milk yield, the gain in weight of the lambs will be promoted.

[Infections with helminths and/or protozoa in cats in animal shelters in the Netherlands]. / Infecties met helminthen en/of protozoën bij katten in asielen in Nederland.

To determine the prevalence of infections with helminths and protozoa in cats in animal shelters, faecal samples from 305 cats from 22 animal shelters in the Netherlands were examined, using a centrifugation-sedimentation-flotation-technique. The association between potential risk factors and the occurrence of an infection was also tested. Infections with helminths and/or protozoa were found in 160 samples (52.5%). Toxocara cati was found in 86 cats (28.2%), Cystoisospora felis in 59 cats (19.3%), Cystoïsospora rivolta in 43 cats (14.1%), Capillaria spp. in 34 cats (11.2%), Ancylostoma tubaeforma in 9 cats (3.0%), Taenia taeniaeformis in 9 cats (3.0%), Aelurostrongylus abstrusus in 8 cats (2.6%), Giardia intestinalis in 3 cats (1.0%), Dipylidium caninum in 2 cats (0.7%) and Toxoplasma gondii in 1 cat (0.3%). The highest prevalence was seen in kittens and stray cats. The main preventive factor against infection was a short stay in a shelter.

[Infections with endoparasites in dogs in Dutch animal shelters]. / Infecties met endoparasieten bij honden in Nederlandse dierenasielen.

Faecal samples from 224 dogs from 23 animal shelters in the Netherlands were examined for endoparasites. In total 20.5% of the faecal sample were positive for helminth and/or protozoa infections. Eggs of Toxocara canis were found in 8.5% of the faecal samples. Other endoparasites found were Toxascaris leonina (0.5%), Trichuris vulpis (4.9%), Uncinaria stenocephala (2.2%), Dipylidium caninum (1.3%), Taenia spp. (0.5%), Cystoïsospora canis (1.3%), and C. ohioensis (1.3%). Dogs younger than 1 year and stray dogs showed the highest prevalence of infection. T. vulpis was found more often in dogs from shelters with a high cleaning frequency.