Albert W. C. A. Cornelissen

Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia canis

Dogs are susceptible to a number of ehrlichial diseases. Among them, canine monocytic ehrlichiosis is an important and potentially fatal disease of dogs caused by the rickettsia Ehrlichia canis. Diagnosis of the disease relies heavily on the detection of antibodies and is usually carried out using the indirect immunofluoresence antibody (IFA) test. The IFA test may be confounded by cross-reactivities between a number of the canine ehrlichial pathogens. This article presents a review of the ehrlichial diseases affecting dogs with reference to their immune responses, host specificities, cross-reactivites and diagnosis. Diagnostic means such as Western immunblot, dot-blot and PCR are discussed. The use of the IFA test as a diagnostic means for E. canis is presented along with its potential pitfalls. The review emphasizes that the disease process, cross-reactivites with other ehrlichial species, multiple tick-borne infections and persistent IFA antibody titers post-treatment, should all be considered when interpreting E. canis serological results.

PROTECTIVE IMMUNITY INDUCED BY VACCINATION WITH TWO HAEMONCHUS CONTORTUS EXCRETORY SECRETORY PROTEINS IN SHEEP

Two excretory secretory (ES) antigens of adult Haemonchus contortus with molecular weights of 15 and 24 kDa, respectively, were evaluated as protective immunogen against haemonchosis. Sheep were vaccinated three times and subsequently challenged with 20 000 infective larvae. Vaccination induced significant reduction (>70%) in mean faecal egg counts and abomasal worm burden compared to the non‐vaccinated control group or adjuvant control group. Vaccination induced ES‐specific antibodies and stimulated infiltration of mast cells in the abomasal tissue.

An observational study of Eimeria species in housed cattle on Dutch dairy farms.

The prevalence of oocysts of Eimeria species in calves (n = 334), yearlings (n = 254) and cows (n = 1314) was determined on 38 Dutch dairy farms. Twelve species of Eimeria were identified in faecal specimens by sucrose-flotation. The prevalences of Eimeria spp. differed markedly in the different age classes on individual farms as well as between farms. The overall prevalence of Eimeria oocysts in faecal specimens was 46% for calves, 43% for yearlings and 16% for cows. The number of oocysts excreted was generally low in cows and yearlings, whereas high numbers of oocysts per gram of faeces (OPG) were exclusively observed in calves. No cases of clinical coccidiosis were observed in this survey. Linear regression analysis showed that there is significant reduction in the OPG levels (P < 0.05) in calves infected with Eimeria, aged between 7 and 38 weeks. Finally, the data are discussed in relation to management practices and the acquisition of immunity.

Induction of protective immunity to Theileria annulata using two major merozoite surface antigens presented by different delivery systems

Allelic forms (Tams1-1 and Tams1-2) of the major merozoite surface antigen gene of Theileria annulata have recently been expressed in Escherichia coli and in Salmonella typhimurium aroA vaccine strain SL3261. To test the potential of subunit vaccines against T. annulata infection, we immunized four groups of three calves with either recombinant (re-) (Tams1-1 and Tams1-2) proteins or naked DNA encoding these antigens. Group I was immunized intramuscularly with both re-proteins incorporated into immunostimulating complexes (ISCOMs). Group II was inoculated intramuscularly with naked plasmid DNA encoding Tams1-1 and Tams1-2. Groups III and IV received S. typhimurium SL3261 [pSTams1-1][pIP5] and SL3261 [pSTams1-2] [pIP5] subcutaneously and orally, respectively. A final group of three animals (Group V) served as an unimmunized control group. Four weeks after the last immunization all calves were challenged with a T. annulata stabilate generated from blood of an infected animal with 30% piroplasm parasitaemia. All calves vaccinated with ISCOMs proved to be protected from T. annulata infection and had generated antibodies against both re-(Tams1-1 and Tams1-2) at the time of challenge. In two of these animals the antibody had a surface binding profile by IFAT. Two of three calves immunized with naked DNA also proved to be protected, but none of the animals had generated any detectable antibodies against the recombinants. Salmonella-based delivery of the recombinants did not induce any protection; two of six animals died of theileriosis and there was no difference between subcutaneous or oral administration. These preliminary results show that re-(Tams1-1 and/or Tams1-2) may elicit protective immune responses in cattle, depending on the antigen delivery system.

Protection studies with recombinant excretory/secretory proteins of Haemonchus contortus.

The efficacy of two recombinant proteins of Haemonchus contortus was studied in both adult sheep and young lambs. These 15 and 24 kDa excretory/secretory proteins were given combined, either supplemented or not with a glycan‐rich insect cell extract. In 9‐month‐old sheep (trial 1), faecal egg output and worm burden were reduced by 49% and 55%, respectively, after vaccination with rec15/24, and by 46% and 65% after vaccination with rec15/24 and glycan extract. No reduction in egg output or number of worms was found in young lambs using the above recombinant proteins plus glycan‐rich extract (trial 2). When trial 1 was repeated (trial 3), the protection could not be reproduced, possibly due to differences in batches of recombinant proteins. In all sheep, independent of their age, rec15/24‐specific immunoglobulin (Ig)G1 and IgA titres were present, but 9‐month‐old protected sheep had significantly higher IgA titres than the lambs. Addition of glycans resulted in lower rec15/24‐specific IgG1 and IgA in 9‐month‐old sheep after challenge. This did not affect the level of protection. A significant negative correlation was found between IgA and worm numbers in protected sheep immunized with rec15/24 supplemented with glycans. Total IgE and rec15/24 specific IgE titres were low. The number of eosinophils, mast cells, sheep mast cell protease (SMCP)+ cells and IgA+ cells did not differ between the protected and unprotected sheep, but the lambs had significantly fewer mast cells independent of their immunization.

Mapping QTL influencing gastrointestinal nematode burden in Dutch Holstein-Friesian dairy cattle.

BackgroundParasitic gastroenteritis caused by nematodes is only second to mastitis in terms of health costs to dairy farmers in developed countries. Sustainable control strategies complementing anthelmintics are desired, including selective breeding for enhanced resistance.Results and ConclusionTo quantify and characterize the genetic contribution to variation in resistance to gastro-intestinal parasites, we measured the heritability of faecal egg and larval counts in the Dutch Holstein-Friesian dairy cattle population. The heritability of faecal egg counts ranged from 7 to 21% and was generally higher than for larval counts. We performed a whole genome scan in 12 paternal half-daughter groups for a total of 768 cows, corresponding to the ~10% most and least infected daughters within each family (selective genotyping). Two genome-wide significant QTL were identified in an across-family analysis, respectively on chromosomes 9 and 19, coinciding with previous findings in orthologous chromosomal regions in sheep. We identified six more suggestive QTL by within-family analysis. An additional 73 informative SNPs were genotyped on chromosome 19 and the ensuing high density map used in a variance component approach to simultaneously exploit linkage and linkage disequilibrium in an initial inconclusive attempt to refine the QTL map position.

Age-related protective immunity after vaccination with Haemonchus contortus excretory/secretory proteins

Protection against an experimental challenge infection by immunization with excretory/secretory products (ES) from Haemonchus contortus, containing predominantly proteins of 15 and 24 kDa, depends on the age of the sheep. Vaccinated sheep 9 and 6 months of age had reduced final worm burdens of 82 and 77, respectively. No reduction in worm burden was found in 3‐month‐old lambs. Nine‐month‐old sheep had significantly higher ES‐specific serum immunoglobulin (Ig)G1 and IgA during immunizations and after challenge infection than 3‐month‐old lambs. There was no correlation within the 9‐month‐old sheep between ES‐specific IgA or IgG1 levels and protection, measured as worm burden. However, when the different age groups were combined, negative correlations between percentage protection and ES‐specific IgA and IgG1 levels after challenge were found. At the end of the experiment, peripheral blood eosinophils and mast cell counts in abomasal tissue were also significantly higher in the vaccinated and challenged 9‐month‐old sheep than in the vaccinated and challenged 3‐month‐old or than in the 9‐month‐old sheep with challenge, but without vaccination. The responses measured in young lambs were similar to the responses in sheep, but the height of these responses was in general of a lower magnitude.

The effect of oxfendazole terminated infections with Haemonchus contortus on the development of immunity in sheep.

The relative contribution of third (L3), fourth (L4) or adult stages of Haemonchus contortus to the development of immunity was evaluated in three groups of sheep subjected to infections terminated by oxfendazole treatments at the L3, L4 or adult stage. A control group did not receive immunising infections. All the groups were challenged with 5000 L3, to evaluate the protection provided by the different protocols. All sheep were necropsied at the end of the experiment to count the abomasal worm burdens. A marked reduction in egg counts after challenge infection was only observed in sheep in which the infection was terminated in the adult stage (Group 4). A significant reduction in worm burden was also observed in Group 4. The immunising infections and/or the challenge infection resulted in moderately elevated IgG antibody levels against L3, L4 and adult somatic antigens in all the groups. In contrast, a strong IgG response against H. contortus excretory/secretory (ES) antigens was observed in the groups in which the immunising infection was terminated in the L4 and the adult stage. An elevated lymphocyte proliferation response against Haemonchus ES antigens was found only in the group that had their immunising infection terminated at the adult stage. The combined data suggest that exposure to and elicited immunological responses to ES antigens are important for the development of immunity against H. contortus.