In vitro stress response to elevated temperature, hydrogen peroxide and mebendazole in Trichinella spiralis muscle larvae.

Three stimuli, elevated temperature, hydrogen peroxide and mebendazole, were compared for their ability to induce heat-shock responses in Trichinella spiralis muscle larvae (L1). In vitro effectiveness of each 'stressor' was evaluated by viability score, protein content and levels of hsp90, hsp70 and hsp60. Detection of the respective heat-shock proteins was done by Western blotting and the heat-shock proteins and quantitation of the immunoblots by image analysis. Exposure of L1 to elevated temperature (e.g. 45 degrees C, 2 h) had no measurable effect. However, exposure to hydrogen peroxide resulted in the induction of constitutive and higher mol. wt heat-shock proteins. In these experiments, heat-shock protein induction correlated strongly with other damage parameters, including loss of viability and increased mortality. Larvae stored in the presence of mebendazole showed no signs of damage. These data indicate that when L1 suffer damage through the action of stimuli, enhancement of heat-shock protein production and damage suffered are causally related.

In vitro shock response to different stressors in free living and pathogenic Acanthamoeba.

Three stresses, viz heat, oxidative and pH shocks, were applied to cultures of three species of Acanthamoeba, free-living Acanthamoeba rhysodes and pathogenic Acanthamoeba castellanii and Acanthamoeba culbertsoni. The effect of each stressor on trophozoite integrity was evaluated by the amount of heat shock protein (HSP)60 and HSP70 produced and by exclusion of 0.2% Congo Red. HSP60 and HSP70 levels were estimated using Western blotting and subsequent densitometric analyses. Unstimulated trophozoites from A. rhysodes produced the lowest background levels of HSP60 and HSP70 and were the amoebae most affected by (mammalian-type) stresses as judged by their enhanced HSP production and decreased viability upon exposure to such conditions. In contrast, unstimulated Acanthamoeba of the pathogenic variety had relatively high background levels of test HSPs and seemed undisturbed by the types of stresses they must deal with when entering their hosts. These studies suggest that high HSP levels in amphizoic acanthamoebae may indicate their involvement in (i) tolerance induction to hosts' stressors and/or (ii) in species' virulence.

Chronic stress in dogs subjected to social and spatial restriction. II. Hormonal and immunological responses.

Two groups of beagles, accustomed to spacious group housing, were subjected to social and spatial restriction and studied for manifestations of chronic stress with a time interval of 7 weeks between the groups. The change from outside group housing (the control period) to individual housing in small indoor kennels resulted in sustained decreases in urinary adrenaline/creatinine and noradrenaline/creatinine ratios for the total group. Urinary dopamine/creatinine and noradrenaline/adrenaline ratios were statistically unaffected. Socially and spatially restricted dogs that had experienced pleasant weather during the control period showed (a) increased salivary and urinary cortisol concentrations, (b) a diminished responsiveness of the pituitary-adrenal axis to a sudden sound blast or exogenous CRH, (c) intact plasma ACTH and cortisol suppressions after dexamethasone administration, and (d) increased concanavalin A induced lymphocyte proliferations. When social and spatial restriction was preceded by a control period during which the weather was bad, these physiological responses were either augmented (lymphocyte proliferation), or offset (salivary and urinary cortisol), or directed oppositely (CRH-induced ACTH and cortisol responses). Together with the previously presented behavioral observations, these data suggest that bad weather conditions during spacious outdoor group housing induced early stress that attenuated the negative appraisal of the subsequent period of social and spatial restriction. In comparison to male dogs, bitches showed increased HPA responses to a sound blast or exogenous CRH. Their increased attenuations of the ACTH and cortisol responses to CRH after 5 weeks of restricted housing indicates that bitches are not only more susceptible to acute stress, but also to chronic housing stress. It is concluded that the quality of circumstances preceding a period of affected well-being determines the magnitude and even the direction of the behavioral and physiological stress responses. Basal salivary and urinary cortisol measurements are useful for the assessment of chronic stress, and of poor welfare in dogs. The use of urinary catecholamine, peripheral leucocyte, and lymphocyte proliferation measures requires further investigation.

A simple method for the demonstration of factors in bovine colostrum capable of causing anaemia in lambs reared free from maedi on bovine colostrum.

Previous studies demonstrated that the anaemia encountered in lambs reared on bovine colostrum and a milk substitute was associated with the presence of immune complexes on lamb erythrocytes. In the present study the usefulness of a panel of 20 sheep sera for the detection of "anti-sheep" factors in bovine colostrum by double immunodiffusion in agarose was investigated. Utilising this method, 353 batches of bovine colostrum have been examined, 132 of which were declared safe for use in the rearing of lambs. When fed to lambs, only 2 samples (1.5%) caused anaemia as compared with up to 20% before this test was introduced. Experiments designed to determine whether the bovine colostra, declared anaemia-prone, would indeed cause anaemia when fed to lambs, showed our method to fully discriminate between safe and unsafe colostra for the rearing of lambs. In a follow-up collaborative study, set up to cover most of the Netherlands, the general validity of the test system described was demonstrated, using 114 batches of safe colostrum to feed 723 lambs. Further experiments are needed to determine the exact nature of the factor(s) involved in this phenomenon.

The significance of reactions to purified fractions of Dermatophagoides pteronyssinus and Dermatophagoides farinae in canine atopic dermatitis.

The significance of reactions to crude extracts and purified fractions of the house dust mites Dermatophagoides pteronyssinus (Der p I and Der p II) and Dermatophagoides farinae (Der f I and Der f II) was evaluated in dogs with clinical manifestations of atopic dermatitis (AD). In 13 healthy control dogs and eight dogs with AD, immediate skin test reactivity was determined to serial dilutions of Der p I, Der p II, Der f I and Der f II. In addition, allergen-specific IgGd antibodies were determined by means of an enzyme-linked immunosorbent assay (ELISA) and Western blots. The results suggest that, in contrast to what occurs in humans and despite immediate skin test reactivity in some dogs, Der p I, Der p II, Der f I and Der f II are unlikely to be major allergens in dogs with AD. However, only serum of atopic dogs consistently binds a 90 kDa polypeptide of D. farinae, as shown by Western blot analysis.

Immunologic aspects of German shepherd dog pyoderma (GSP).

In 21 dogs with clinical features of German Shepherd dog Pyoderma (GSP) parameters of the specific and aspecific immune system have been examined. Chemotaxis and killing capacities of neutrophilic leucocytes were undisturbed, whereas in skin biopsies no specific immunoglobulin or complement deposits were found with immunofluorescence. With double immunodiffusion, antibodies against Gram-positive bacteria were found. In a laser nephelometric assay significantly elevated levels of IgG, IgGab, IgGd, IgM and bacterial components, associated and non-associated with circulating immune complexes, were detected. However, no relation was found with the disease state. It is concluded that dogs with GSP are immunologically normal reactors. A bacterial hypersensitivity reaction is hypothesized as a possible initiating factor in the pathogenesis of GSP.

Antibodies to immunoglobulin-G in dog sera, synovial fluids and aqueous humor: a comparative study of rheumatoid factor assays, suitable for routine application.

The incidence of anti-IgG antibodies (rheumatoid factors, RF) in body fluids (sera, synovial fluids and aqueous humor) selected from 62 normal and 275 diseased dogs was studied. Fluids were assayed by canine versions of standard agglutinating and/or precipitating RF assays with routine application in human practice. The number of RF detected by dog IgG-coated particles was substantially higher by latex fixation test (LFT) than by modified Rose-Waaler (RW) test (61/144 vs. 14/144). This did not result from false positives by LFT since latex activity was completely inhibited by aggregated dog IgG. Some evidence is presented indicating that results obtained by standard RW in particular, but also those obtained by standard LFT, might be improved by modifying testing conditions currently used. Body fluids were further studied for the presence of precipitins to aggregated dog IgG in 0.6% agarose (gel precipitation test (GPT]. The frequency of RF was higher by GPT than by LFT, both in normal control fluids (for sera 26/52 vs. 19/52) and patient material (for sera 135/197 vs. 95/197). Thus, the canine RF appear to be a serum component with an unexpectedly high frequency in both normal and diseased dogs, but grossly underestimated by the recommended routine RF assays based on agglutination. The GPT, which combines a superior detection rate of theoretically also agglutinating RF with an inability to detect RF quantitatively, seems an ideal RF 'indicator' test to dictate improvements to the quantitative LFT/RW assays so as to facilitate RF detection at clinically relevant concentrations. Thus optimized, RW/LFT would provide the optimal detection apparatus for the ultimate isolation of the relevant 'RF' repertoire present, for comparative studies aimed ultimately at unraveling the etiopathogenesis of the 'real' RF.

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.

Antigen-specific immune responses in cattle with inherited beta2-integrin deficiency.

The significance of beta2-integrins for the generation of antigen-specific immune responses in vivo was studied employing the bovine model of beta2-integrin deficiency. To that end four cattle with bovine leukocyte adhesion deficiency (BLAD) and healthy age-matched controls were immunized with tetanus toxoid (TT) and rabies virus (RV) vaccines three times in monthly intervals. In addition, two animals with BLAD and three controls received a fourth vaccination 8 months after the start of the study. Proliferative responses of peripheral blood mononuclear cells (PBMC) to the antigens TT and RV as well as specific serum immunoglobulin G (IgG) titers were determined in intervals for up to 10 months after primary vaccination. Proliferative responses of PBMC to TT and RV were substantially lower in cattle with BLAD than in controls, although PBMC from cattle with BLAD were shown to have the capacity to proliferate in the response to the mitogen concanavalin A. Occurrence of antigen-specific IgG titers was delayed and they were considerably lower in cattle with BLAD compared to controls. Finally, treatment of TT- and RV-stimulated PBMC from an immunized control with different concentrations of the anti-CD18 monoclonal antibody R15.7 resulted in a dose-dependent inhibition of lymphocyte proliferation to almost 100%. The results of the present study show that beta2-integrin deficiency leads to delayedand severely impaired immune responsiveness in vivo. The observations that antibody production, although considerably delayed and impaired, does occur and that apparently class-switching takes place in BLAD indicate T-cell reactivity in vivo.

Allograft rejection in cattle with bovine leukocyte adhesion deficiency.

In the present investigation cell-mediated immunity in animals with bovine leukocyte adhesion deficiency (BLAD) was studied by means of skin transplantation experiments. Autograft and allograft behaviour in animals with BLAD was compared with the behaviour of simultaneously transplanted autografts and allografts in healthy controls. Allograft survival time was prolonged in three BLAD cattle (28, 30, and 72 days) compared to six healthy controls (12-14 days). When transplantations were repeated on one animal with BLAD using skin grafts from the same donor, accelerated rejection was observed (allograft survival time decreased from 72 days at primary to 35 days at secondary and to 21 days at tertiary transplantation), suggesting the development of immunological memory. Graft-infiltrating lymphocytes that were obtained from allograft biopsies during the period of rejection, were shown to be from recipient origin (beta 2-integrin negative). Our findings demonstrate that, although prolonged allograft survival is observed in cattle with BLAD, skin allografts are ultimately rejected.

Detection of canine cytokine gene expression by reverse transcription-polymerase chain reaction.

Further characterization of the canine immune system will greatly benefit from the availability of tools to detect canine cytokines. Our interest concerns the study on the role of cytokines in canine visceral leishmaniasis. For this purpose, we have designed specific primers using previously published sequences for the detection of canine IL-2, IFN-gamma and IL10 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). For IL-4, we have cloned and sequenced this cytokine gene, and developed canine-specific primers. To control for sample-to-sample variation in the quantity of mRNA and variation in the RT and PCR reactions, the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a housekeeping gene, were determined in parallel. Primers to amplify G3PDH were designed from consensus sequences obtained from the Genbank database. The mRNA levels of the cytokines mentioned here were detected from ConA-stimulated peripheral mononuclear cells derived from Leishmania-infected dogs. A different pattern of cytokine production among infected animals was found.

Leukocyte adhesion deficiency in a Dutch Holstein calf: a case with a clear-cut family history.

A leukocyte adhesion deficiency characterized by recurrent (predominantly bacterial) infections, lack of extravascular polymorphonuclear leukocyte (PMN) and pus formation has been described first in humans and then in dogs, and recently also in cattle. Because of important clinical similarities, a unitary explanation for the leukocyte adhesion deficiency (LAD) syndrome in mammals is proposed, inasmuch that an intrinsic leukocyte defect (i.e. mutations in genes encoding the common CD18 subunit), is thought to cause the disease. However, thus far, the hallmark of such intrinsic leukocyte defects, notably their heritability (or familial incidence), has not (yet) been unequivocally demonstrated. This is the first report to describe the occurrence of four Dutch bovine LAD (BLAD) cases with the clearest familial clustering observed to date. The diagnosis was based on the clinical features of very poor thriving, in general, of the calves, hyperneutrocytosis without appreciable left shift, and the absence of PMN CD11a, or CD11b, or CD11c using monoclonal antibodies (mAb) and/or Concanavalin A binding activity of PMN lysates in immunoblots. Interestingly, a familial clustering was observed also for below-normal PMN CD11c expression. Thus, a cow with low CD11c expression (50.4%) and delivering three of the study BLAD calves, also had a healthy descendant with low (44.9%) PMN CD11c expression. These findings suggested the possibility that both subnormal expression and lack of PMN CD11 expression are inheritable factors in cattle. Furthermore, a large prospective study using the present mAb for selecting relatives expressing the complete spectrum (0 to > or = 90%) of PMN CD11/CD18 expression would create a comprehensive study population for understanding both the role of genetic factors and of survival strategies in BLAD.

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.

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.

Using heat shock proteins as indicators of the immune function in wistar rats during a secondary Trichinella spiralis infection.

The muscle, liver, brain and spleen tissues from Wistar rats with either a primary Trichinella spiralis infection alone, or reinfected 45 days after primary infection, were collected at Days 1, 7, 14, 20 and 27 post reinfection. They were then assayed for levels of four heat shock proteins (HSPs), i.e. hsp90, hsp70, hsp60 and hsp25. Detection and quantitation of the separate HSPs in tissue specimens were achieved using Western blot and image analysis technique, respectively. The results show that the elements affecting altered expression of rat organs' HSP were 'neutralized' by resistance-related events in immunized rats. Thus, while rat organs exhibited varying HSP levels in primary infections [Martinez, J., Perez-Serrano, J., Bernadina, W., Rodriguez-Caabeiro, F., 1999. Parasitology 118, 202-209], there was, in reinfected versus primary-infected rats, no difference in test HSPs levels in any organ, and at any time within the time course of this study. We have interpreted the results by using the model that involves induction of anti-T.spiralis immunity during primary infection and (almost) complete removal of effectors of tissue injury (infective T.spiralis larvae and newborn larvae) during reinfection.

Purification and preliminary characterization of a protease from the excretion-secretion products of Trichinella spiralis muscle-stage larvae.

A protease from excretion-secretion products of Trichinella spiralis muscle-stage larvae was purified by continuous elution electrophoresis. The state of purification was analyzed electrophoretically using one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme was shown to be a single polypeptide with an estimated molecular mass of 35 kDa and isoelectric point of 6.2. Following purification, the enzyme activity was measured by hydrolysis of gelatin, azocoll, azoalbumin, azocasein and collagen as substrates. Maximal azocollytic activity was at pH 5 and a temperature of 37 degrees C. Finally, the proteolytic activity was partially inhibited by N-alpha-p-tosyl-lysine chloromethyl ketone, chymostatin and E-64.

Detection of heat shock protein-70 from Trichinella spiralis larvae using a modification of the routine western blotting procedure.

This is the first study that establishes a standardized western blotting method for the detection of heat shock protein (HSP) 70 from Trichinella spiralis using (selected) monoclonal antibodies (mAbs). Enhancement of HSP transfer onto the supportive membrane and increased retention of protein by the membrane are prominent features of the procedure. The reactivity of T. spiralis HSP70 on western blots was substantially increased by the use of a 10% acrylamide gel, the optimization of conditions during electrotransfer, and transfer onto Immobilon membranes. These data indicate that mAbs actually capable of detecting the agent of interest may be discarded because of nonoptimal testing conditions. We suggest that this method will aid in understanding the role and function of T. spiralis HSP70 in host-parasite (inter)relationship(s).

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.

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.

Anoplocephalid cestodes of veterinary and medical significance: a review.

Cestodes of the family Anoplocephalidae Cholodkovsky, 1902, in their adult form, parasitize a variety of hosts, including reptiles, birds and mammals. To complete their life cycle, an intermediate host is required. This study gives a critical review of the life cycles of genera principally important to veterinary medicine (but sporadically infecting man): Anoplocephalinae (Anoplocephala, Anoplocephaloides, Bertiella and Moniezia) and Thysanosomatinae (Avitellina, Stilesia, Thysaniezia and Thysanosoma), using data reported by others and our own observations. The accepted paradigm on the biology of the anoplocephalid cestodes is that oribatid mites (Acarina) serve as intermediate hosts. However, as regards the genera Avitellina, Thysaniezia and Thysanosoma, it is still unclear whether oribatid mites are indeed the intermediate hosts, as larval forms (cysticercoids) have also been found in collembolans and psocids. Using the controversial biological cycle of Thysanosoma actinioides (Diesing, 1834), a theoretical methodological research proposal for parasitology was constructed which attempts to define a conceptional mark enabling us to predict and explain the parasite-hosts' related phenomenon. Aspects of this proposal are discussed using the biology of the cestodes of family Anoplocephalidae, as examples.