W.E. Bernadina

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.

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.

An immunodiffusion assay for the detection of canine leishmaniasis due to infection with Leishmania infantum.

An immunodiffusion assay (IDA) with polyethylene glycol (PEG) was tested for usefulness as diagnostic test for canine leishmaniasis (CL). A comparative analysis of dog sera was made using IDA with PEG, immunofluorescence assay (IFA) and enzyme immunosorbent assay (ELISA) techniques. Fourty-four dogs from Italy with CL (endemic dogs) and eight Dutch dogs with CL contracted in South Europe (expatriate dogs) were tested together with 40 endemic and 35 expatriate controls. Specificity did not differ substantially among the serotests, ELISA in endemic dogs being the least specific (mean specificity given in IFA, IDA and ELISA, 100%, 98% and 93.5%, respectively). Sensitivity in expatriate dogs was 100% for all serotests but was highly variable in endemic dogs. In parasite-negative dogs, IFA had the most sensitivity, i.e., 80.5% compared to 69% for both ELISA and IDA. In contrast, ELISA in parasite-positive endemic dogs had a sensitivity of 100% whereas both IFA and IDA gave a sensitivity of 93%. Despite its slightly lesser sensitivity than IFA or ELISA (2-6% and 5% respectively) in endemically infected dogs, IDA with PEG method may help to bring the diagnosis of CL within reach of the veterinary practitioner.

BIOTIN-AVIDIN AMPLIFIED ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE MEASUREMENT OF CANINE SERUM IGA, IGG AND IGM

An amplified capture enzyme-linked immunosorbent assay (ELISA) has been developed by the use of the biotin-avidin detection system, for the measurement of canine plasma immunoglobulins (Ig) A, G and M. Test responses of dilutions of both the Ig standards and test plasma samples were consistently linear (r > 0.987) for the three Ig classes. The within-assay variation was 3.53 per cent for IgG, 5.84 per cent for IgM and 6.34 per cent for IgA. The analytical recoveries were 95 per cent for IgA, 97 per cent for IgG and 98 per cent for IgM. The lower detection limits of the assay were 38.4 ng ml-1 for IgG, 20.3 ng ml-1 for IgM and 41.2 ng ml-1 for IgA. The results indicate that this ELISA has a much higher sensitivity than the single radial immunodiffusion assay or the non-amplified ELISA for measurements of canine Igs, but has a comparable specificity and precision.

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.

Heat shock protein synthesis over time in infective Trichinella spiralis larvae raised in suboptimal culture conditions

Changes in the viability, infectivity and heat shock protein (Hsp) levels are reported in Trichinella spiralis first stage larvae (L1) stored in 199 medium for up to seven days at 37 degrees C. These conditions induce stress that the larvae, eventually, cannot overcome. After three days of storage, the infectivity and viability were unchanged, although higher Hsp70 levels were observed. After this time, larvae gradually lost viability and infectivity, coinciding with a decrease in Hsp70 and Hsp90 and an increase in actin (a housekeeping protein). In addition, a possibly inducible heat shock protein, Hsp90i, appeared as constitutive Hsp90 disappeared. No significant changes in Hsp60 levels were detected at any time. These results suggest that heat shock proteins initially try to maintain homeostasis, but on failing, may be involved in cell death.

Expression of Hsp90, Hsp70 and Hsp60 in Trichinella species exposed to oxidative shock

Stress response and phosphorylation of heat shock proteins (HSPs) 60, 70 and 90 were studied in Trichinella nativa, T. nelsoni, T. pseudospiralis and T. spiralis larvae at 30-min intervals following exposure to 20, 100 and 200 mM H2O2. There was a time- and dose-dependent differential survival for the infective stage larvae (L1) of these four Trichinella species. Immunoblotting analysis revealed that constitutive Hsp60 and Hsp70, but not Hsp90, from test Trichinella species are constitutively phosphorylated on serine/threonine residues as they converted to forms with increased sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility by treatment with alkaline phosphatase. After exposure to H2O2, while there was a time-related occurrence of the three HSPs with decreased SDS-PAGE mobility, these HSPs were insensitive to alkaline phosphatase except in the case of exposure to 20 mM H2O2 for Hsp60 from all Trichinella species and Hsp70 from T. spiralis and T. nelsoni. The synthesis of HSPs forms with decreased SDS-PAGE mobility is a susceptibility signal because the lower concentration of peroxide (20 mM) did not cause a decrease on HSPs SDS-PAGE mobility in T. spiralis and T. nelsoni, the two more resistant selected Trichinella species.

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.

Prior immunity to Trichinella spiralis prevents (re)occurrence of an explicit stress response in intestines but not in mesenteric lymph nodes, heart and lungs from reinfected rats.

We recently showed that, in our Trichinella spiralis rat model, first exposure, but not re-exposure to infective-stage larvae evoked heat shock responses in 4 test organs. Our work, however, failed to implicate either early complete clearance of challenge muscle larvae (ML), or rapid elimination of newborn larvae (NBL) in the phenomenon noted in reinfected rats. This study clarifies that issue using 2 established facts in T. spiralis biology and anti-T. spiralis immunology. That is, adult worms injure gut cells and immune destruction of NBL requires release of material also toxic to host cells. To approach the above problem we analysed relevant and irrelevant rat organs for increased heat shock protein (HSP) production at 1, 7, 14, 20 and 27 p.i. during first and second infections. Organs examined were intestines, mesenteric lymph nodes (MLN), heart and lungs. Using densitometric analyses of immunoblots, increased HSP expression was detected on day 7 in intestines from both primary and secondary-infected rats albeit that the change in the latter was just short of significant. Interestingly, MLN only exhibited increased HSP levels in the reinfected rat model with increased HSP levels persisting for 1 week. A lasting shock response was detected in reinfected rats; in contrast, first exposure resulted in shock responses being evident in lungs at either day 7 or day 14, only. These findings suggest that (i) in immune rats, a few challenge ML develop into adults, produce NBL which are trapped within MLN, and (ii) that anti-T. spiralis and/or anti-NBL immunity is associated with an, as yet, uncomprehended stress to hosts heart tissues.