Maternal immunity enhances systemic recall immune responses upon oral immunization of piglets with F4 fimbriae

F4 enterotoxigenic Escherichia coli (ETEC) cause diarrhoea and mortality in piglets leading to severe economic losses. Oral immunization of piglets with F4 fimbriae induces a protective intestinal immune response evidenced by an F4-specific serum and intestinal IgA response. However, successful oral immunization of pigs with F4 fimbriae in the presence of maternal immunity has not been demonstrated yet. In the present study we aimed to evaluate the effect of maternal immunity on the induction of a systemic immune response upon oral immunization of piglets. Whereas F4-specific IgG and IgA could be induced by oral immunization of pigs without maternal antibodies and by intramuscular immunization of pigs with maternal antibodies, no such response was seen in the orally immunized animals with maternal antibodies. Since maternal antibodies can mask an antibody response, we also looked by ELIspot assays for circulating F4-specific antibody secreting cells (ASCs). Enumerating the F4-specific ASCs within the circulating peripheral blood mononuclear cells, and the number of F4-specific IgA ASCs within the circulating IgA⁺ B-cells revealed an F4-specific immune response in the orally immunized animals with maternal antibodies. Interestingly, results suggest a more robust IgA booster response by oral immunization of pigs with than without maternal antibodies. These results demonstrate that oral immunization of piglets with F4-specific maternal antibodies is feasible and that these maternal antibodies seem to enhance the secondary systemic immune response. Furthermore, our ELIspot assay on enriched IgA⁺ B-cells could be used as a screening procedure to optimize mucosal immunization protocols in pigs with maternal immunity.     

Immune response to an Actinobacillus pleuropneumoniae vaccine in swine

Piglets vaccinated with an inactivated tetravalent vaccine (Pleurovac 4) against Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7, produced circulating antibodies after a first intramuscular injection and showed an anamnestic reaction after a second. The rise in antibody levels in vaccinated piglets was statistically significant when compared with those of the control group. The administration of 1 or 2 mL of vaccine did not lead to significantly different antibody levels. The specificity of the immune response is demonstrated by an increase in titer to all four serotypes in pigs given the tetravalent vaccine, but an increase in titer to only two serotypes in pigs given a bivalent vaccine (Pleurovac).     

Laboratory diagnosis of human toxocariasis

Toxocariasis is a helminth zoonosis caused by infection with the larvae of Toxocara spp. ascarid worms. Only two species, Toxocara canis and Toxocara cati, are recognised as causative agents of human disease. The best choice for serodiagnosis of the generalised forms of toxocariasis, visceral larva migrans (VLM) or covert toxocariasis, relies upon the initial use of TES-ELISA, after which any positive result should subsequently be tested by Western blotting (WB). Covert toxocariasis is mostly a benign infection, so a large majority of infected subjects are asymptomatic or have very few symptoms and therefore go undiagnosed. In this form, this helminthosis is often self-limiting, leaving residual specific antibodies. A positive serodiagnosis caused by residual antibodies that do not have any diagnostic significance can be associated with any infectious or non-infectious disease. If separated from the ongoing clinical and laboratory context, such a positive result has no diagnostic value and should be only taken into account after the possible etiologies of any observed syndromes have been ruled out. Unlike the methods used for the immunodiagnosis of bacterial, viral or protozoal (toxoplasmosis) infections, it is not possible with toxocariasis to assess the age of the presence of specific IgG using the levels of specific IgM because IgM antibodies can be found throughout the course of helminthiasis. The detection of other classes of immunoglobulins, namely IgE and IgA, the subclasses, namely IgG4 or circulating Ag was proven to be unable to discriminate between active and self-cured generalised toxocaral infections. Currently, the diagnosis of an active covert toxocariasis relies upon indirect arguments, e.g., the presence of otherwise unexplained symptoms along with blood eosinophilia and/or elevated levels of eosinophil cationic protein (ECP). This situation is far from ideal and more research should be carried out to solve this difficult problem.     

Potential viral vectors for the stimulation of mucosal antibody responses against enteric viral antigens in pigs

Four viruses were compared for their ability to induce an intestinal antibody response in piglets. Antibodies were not detected in response to oral vaccination with either fowlpox virus or a baculovirus (BV). Simultaneous oral dosing and parenteral inoculation with high concentrations of BV in an oil emulsion adjuvant induced high levels of circulating virus neutralising (VN) antibodies, and also low levels of intestinal antibodies when booster doses of virus were given. In response to oral vaccination with swinepox virus (SPV), low levels of circulating and intestinal VN antibodies, and higher titres of antibodies reactive in an enzyme immunoassay, including intestinal antibodies of the IgA class, were detected. Oral vaccination with porcine adenovirus type 3 (PAV-3) stimulated both circulating and intestinal VN antibodies, and IgA antibodies were demonstrated in the intestinal contents. It was concluded that SPV and PAV-3 might be suitable vectors for the expression of genes encoding the protective antigens of porcine enteric viruses.     

Immunologic systems and protection in infections caused by intracellular blood protista

Rickettsiaea and protozoa which invade circulating leukocytes and erythrocytes are discussed in this communication. Characteristic features of animal diseases caused by these agents are (1) they are transmitted by arthropod vectors, (b) most cause devastating losses to the livestock industry, and (c) some are important to human health. A common characteristic of blood protista is their ability to survive for a long time in immunologically hostile hosts. This can be attributed to their intracellular domicile, possible incorporation of host cell proteins in their structure, and antigenic variations causing evasion of the host's surveillance mechanism. The last decade has seen an expansion in the application of immunologic principles to the study of blood protista. Infections caused by blood protista stimulate humoral and cell-mediated immune responses. Antibodies have been measured in serodiagnostic procedures. Cell-mediated immunity in combination with antibodies of various activities seem to be the main protective mechanism. A vaccine using a live attenuated agent (Anaplasma marginale) which induces and maintains the above responses constitutes a novel, realistic, and practical approach to control of blood protistan diseases.     

Effect of substrate selection on indirect immunofluorescence testing of canine autoimmune subepidermal blistering diseases

The detection by indirect immunofluorescence (IIF) of circulating antibodies in the serum of dogs with autoimmune subepidermal blistering diseases (AISBD) was regarded for a long time as an unrewarding tool. It was, however, demonstrated in humans that the sensitivity of IIF assays depended on the selection of the substrates used. The effects of substrate selection on IIF tests was thus studied by examining sera from 12 dogs with AISBD tested against 8 different substrates from 3 different normal dogs. Patients with AISBD suffered from bullous pemphigoid (n = 4 sera), mucous membrane pemphigoid (n = 4 sera), and epidermolysis bullosa acquisita (n = 4 sera). Substrates included canine tongue, canine lip, canine dorsal haired skin, and ventral haired skin. The same 4 substrates were also split with salt splitting technique (using 1 M sodium chloride), in order to cleave the basement membrane within the lamina lucida and to expose the targeted antigens. The strength of the specific fluorescence of each slide was scored after processing for IIF testing with anti-canine IgG polyclonal antibody. Other criteria, such as background fluorescence, easiness of the interpretation, and variations within a same substrate, were also assessed. Intact canine lip and canine salt-split lip demonstrated consistently stronger intensity of fluorescence and a better ease of interpretation. We concluded that the performance of IIF tests with such substrates was a reliable tool for the detection of circulating IgG autoantibodies of canine patients with AISBD.     

Effect of florfenicol on the immune response of rainbow trout (Oncorhynchus mykiss)

Florfenicol, a drug effective against several bacterial diseases of fish, was tested for possible immunomodulatory effects. The aim of the study was to follow the kinetics of the immune response after vaccination with simultaneous oral antibiotic treatment. The fish were immunised with a commercial oil-based divalent (furunculosis/vibriosis) vaccine and were simultaneously given oral antibiotic treatment. The specific immune response was monitored by analysing the levels of specific antibodies with ELISA. As an indicator of the non-specific immune response the phagocytic activity of circulating leucocytes was measured by a chemiluminescence assay. Total circulating leucocyte counts and differentials were also monitored. The disease resistance was evaluated by challenge tests at the end of the experiment. The results showed that florfenicol did not have any significant effect on antibody production and circulating leucocyte levels but caused a suppression in chemiluminescence response/phagocytic cell 5-6 weeks after vaccination. The survival after challenge was slightly suppressed by the florfenicol treatment. The RPS-value for the vaccinated group was 98% and for the florfenicol-treated group was 88%.     

Evaluation of circulating PD-1 and PD-L1 as diagnostic biomarkers in dogs with tumors

BackgroundProgrammed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) have important roles in tumor evasion of the immune system.ObjectivesThis study aimed to assess the diagnostic utility of circulating PD-1 and PD-L1 levels in healthy dogs and dogs with tumors.MethodsCirculating PD-1 and PD-L1 levels in the serum of 71 dogs with tumors were compared with those of 52 healthy dogs by performing enzyme-linked immunosorbent assay (ELISA).ResultsThe ELISA results revealed higher circulating PD-1 and PD-L1 levels in dogs with tumors (2.9 [2.2–3.7] ng/mL; median [IQR] and 2.4 [1.4–4.4] ng/mL, respectively) than in healthy dogs (2.4 [1.9–3.0] ng/mL; p = 0.012 and 1.4 [0.9–2.1] ng/mL; p < 0.001, respectively). Especially, there was a significant difference in circulating PD-1 levels between healthy dogs and dogs with malignant epithelial tumors (2.4 [1.9–3.0] ng/mL and 3.1 [2.6–4.4] ng/mL, respectively; p < 0.01). In addition, there was a significant difference in circulating PD-L1 levels between healthy dogs and dogs with lymphomas (1.4 [0.9–2.1] ng/mL and 2.7 [1.6–5.8] ng/mL, respectively; p < 0.001).ConclusionThis study indicates that circulating PD-1 and PD-L1 have potential as tumor diagnostic biomarkers in dogs with tumors.     

Comparison of monoclonal and polyclonal antibodies for the detection of canine IgG1 and IgG2, and associations with infection outcome in Leishmania infantum naturally infected dogs

In murine models of leishmaniasis, IgG subclass expression is a proxy measure for Th1/Th2 cellular immune response bias. However, in dogs, the reservoir of zoonotic visceral leishmaniasis, no consistent association has been described between IgG subclass ratios and disease resistance. Inconsistent results may reflect lack of specificity of commonly used commercial antibodies. Our aim was to measure IgG1 and IgG2 responses to crude Leishmania antigen using commercial polyclonal antibodies for comparison with a panel of commercially unavailable monoclonal antibodies, in a cohort of 60 naturally infected dogs, and to compare associations between subclass responses and clinical or parasitological outcomes. IgG1 and IgG2, measured by both antibodies, were higher in clinically symptomatic than in asymptomatic dogs (P ≤ 0.03), reflecting general upregulation of IgG in infected dogs. Unlike the murine model, canine IgG2:IgG1 ratios were not predictive of clinical or parasitological outcomes of infection. Associations between subclass levels and positivity by bone marrow culture and PCR were not consistent when measured with different antibodies. Further research is needed to re-evaluate the specificity of commercially available IgG subclass antibodies.     

Determination and Occurrence of Histamine in Rumen Liquor of Sheep

Methods for the determination of histamine in rumen liquor and different feeds are described. The procedures involve adsorption of histamine on Amberlite IRC-50 columns and biological determination of the histamine content of eluates from the ion-exchange resin. By these methods 70—80 % of added histamine were recovered and histamine concentrations as low as 0.01 μg histamine diphosphate per ml of rumen liquor could usually be estimated.Occurrence and formation of histamine in rumen contents of sheep have been examined with the following results:

1. Feeding invariably increased the concentration of histamine in the rumen, the increments being related to the histamine content of the feed.
2. A small formation of histamine seemed to take place in rumen content both in vivo and in vitro after feeding hay or concentrates.
3. The in vitro formation of histamine in rumen contents was greatly increased when L-histidine (10 mg/ml) was added. The formation of histamine varied widely from day to day and was further related to the time of feeding.
4. Pyridoxal-5-phosphate increased the in vitro formation of histamine in rumen content.

     

Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.     

Effects of somatostatin immunoneutralization on growth and endocrine parameters in chickens

The effects of somatostatin immunoneutralization on growth rate, growth hormone (GH) secretion and circulating insulin-like growth factor I (IGF-I) concentrations were investigated in chickens through the use of passive and active immunization techniques. Intravenous bolus injection of goat-antisomatostatin stimulated a significant (P less than .05) increase in plasma GH levels for one hour post-injection in four and six week old male broiler chickens. The GH response to an intravenous bolus injection of hGRF44NH2 was similar in the antisomatostatin treated chicks and normal goat serum treated controls. Despite the presence of circulating somatostatin antisera after 28 hours, plasma GH levels were not different between control and antisomatostatin-treated chicks at that time. Continuous administration of somatostatin antisera by Alzet pump over a two-week period resulted in significant (P less than .05) elevations in plasma GH levels at one week post-implantation and in circulating IGF-I concentrations after two weeks of administration. Chicks which developed antibodies against somatostatin following active immunization exhibited a 7.1% increase in growth rate which was associated with a significant decrease in abdominal fat. However, neither GH nor IGF-I concentrations were elevated in the chicks which developed somatostatin antibodies. Thus, the benefits gained from somatostatin immunoneutralization may be exerted through mechanisms other than GH.     

Differential activation of mast cells by antigens from Trichinella spiralis muscle larvae,adults, and newborn larvae

Mast cell (MC) hyperplasia and activation are prominent features in Trichinella spiralis infection. Indeed a temporal correlation has been shown between the kinetics of intestinal mastocytosis, release of inflammatory mediators from MC, and adult worm loss, which constitutes a major component of the defense against T. spiralis infection. It is well known that during the intestinal phase of trichinellosis, muscle larvae (ML) and adult worms (AD) enter into contact with the host; however, interaction with MC may also occur during migration of newborn larvae (NBL). Therefore, it is plausible that antigens from these developmental stages could activate MC. We have previously demonstrated by in vitro assays that T. spiralis muscle larval (TSL-1) antigens activate MC through an Ig-independent mechanism leading to the release of histamine, MC protease 5, IL-4 and TNF alpha. In this work we evaluated whether total antigens from AD or NBL could activate unsensitized MC and we compared this activation with the activation seen when MC are stimulated with TSL-1 antigens. MC activation was also tested with affinity chromatography purified antigens from NBL using the monoclonal antibody CE-4 that recognizes NBL surface components. The results obtained in this study showed that AD total extracts and TSL-1 antigens induced the release of histamine but not β-hexosaminidase from unsensitized MC, suggesting a selective secretion of MC mediators. In contrast, NBL total extracts or purified NBL antigens did not induce the release of either histamine or β-hexosaminidase from MC. Interestingly, AD and ML are the stages that interact with the host during the intestinal phase of infection. The mechanisms involved in TSL-1 and AD activation of unsensitized MC may function together with other mechanisms of MC activation in host protection against T. spiralis.     

Variations in the immune response to natural Schistosoma mattheei infections in calves born to infected mothers

During previous work Schistosoma antibodies and circulating antigens were detected at birth in the serum from some calves born to Schistosoma mattheei infected mothers. The objectives of the present survey were: (1) to investigate the proportion of calves, born to cows infected with S. mattheei, which have specific antibodies and circulating schistosome antigens present in their serum at birth and (2) to investigate whether the presence or absence of these specific antibodies and/or circulating antigens at birth may affect the pattern of a natural S. mattheei infection in calves from 4 to 5 months of age, when the colostral antibodies are thought to be of negligible importance. A total of 28 calves born to infected mothers were randomly selected. Faeces, serum and colostrum samples were collected from the cows at calving, serum samples were collected from the calves at birth (day 0), after intake of colostrum (day 1) and monthly thereafter up to the age of 10 months. Both serum and colostrum samples were analysed for IgG(H+L) against SWAP mattheei and schistosome circulating anodic antigen (CAA) levels. The calves were exposed to a natural challenge from the age of 4-5 months. Faecal samples were collected from the calves monthly, starting at an age of 5 months up to 10 months, and were examined for faecal egg counts. Nine (group 1) out of the 28 calves were found to have specific antibodies in their serum at birth, in 5 of them CAA levels were also detected. In the other 19 calves (group 2) no IgG(H+L) or CAA were detected. At the end of the study faecal egg counts and CAA levels were significantly lower in calves from group 1 compared to group 2. Results confirm earlier work that specific antibodies and circulating antigens may be present in serum from calves at birth, and show that these calves have lower faecal egg counts and CAA levels after exposure to a natural challenge.     

The effect of medication with metichlorpindol and methylbenzoquate on the development of antibodies as measured by the fluorescent antibody (IFA)_test against Eimeria tenella and Eimeria maxima

The development of antibodies as measured by the indirect fluorescent antibody (IFA)_ test was studied in groups of birds infected with Eimeria tenella or E. maxima and fed diets containing methylzoquate (MBQ) and/or meticlorpindol (MCP). The drugs suppressed both the E. tenella and E. maxima infections, the extent depending on the concentrations in the feed. The IFA test titres in both the E. tenella and E. maxima infections were related to the oocyst production. Effective suppression of the infection resulted in on or minimal IFA response. Since both drugs used inhibit the sporozoites, it is concluded that development of the parasite is necessary to stimulate the host to produce circulating fluorescent antibodies.     

Training Program Intensity Induces an Acute Phase Response in Clinically Healthy Horses

Physiological and hematochemical changes associated with exercise have been extensively investigated in equine species. It is known that stress elevates circulating levels of acute phase proteins (APPs). This survey evaluated whether horses trained with different training programs exhibit changes in APP levels after exercise event. Twenty Saddle Italian horses (11 geldings and 9 females, 9 ± 1 years old, body weight of 425 ± 35 kg) were divided into two equal groups according to the intensity of training programs they were subjected: group A was subjected to an intense training program, group B was subjected to a moderate training program. At the end of the training period, horses were subjected to a simulated exercise event (show jumping course of 400 m length with 12 obstacles). From horses, blood samples were collected at rest conditions (T_REST) and after 12 and 24 hour from the end of exercise (T_{12 h} and T_{24 h}); the concentration of serum amyloid A (SAA), haptoglobin, albumin, total proteins, iron, and fibrinogen was assessed. The circulating levels of SAA, fibrinogen, and iron were influenced by simulated exercise event (P < .01), starting from 12 hour after the end of exercise, suggesting the onset of an acute phase–like response, and it would seem that training program intensity the horses underwent also affected the degree of response, although only SAA values were significantly different between groups (P < .001). The findings obtained suggest that jumping exercise induces an acute phase response; however, further studies are advocated to better evaluate mechanisms by which exercise activates this response in the athletic horse.     

A novel approach to growth promotion using auto-immunisation against somatostatin. I. Effects on growth and hormone levels in lambs

Nine three-week-old lambs were immunised against somatostatin linked to human serum globulin. Significant antibody titres were obtained in all these lambs. The rate of weight gain was greater in those animals immunised against somatostatin (treated) than in the control animals which were immunised against globulin alone (P < 0.001). The final height of the treated lambs was also significantly greater (P < 0.05) as a result of increased height velocity during the period of treatment (P < 0.01).Evaluation of the animals both before and after slaughter indicated that there was no effect of treatment upon body composition, but rather that there had been proportional growth. The carcass assessments revealed that the treated lambs had a longer leg-length (P < 0.01) and had a greater warm carcass weight (P < 0.05) than the control animals, but had similar scores for fat and muscle covering. There was no difference in wool weight between the two groups.No significant difference was found between the groups in the basal levels of either insulin or growth hormone. However, growth hormone release following arginine infusion was greater in the treated animals. There was also a small, but significant, difference (P < 0.05) in the levels of bioassayable somatomedin activity between the treated and control lambs, the treated lambs having higher somatomedin activity.Histological examination of the pancreas from control and treated lambs revealed an increased incidence of abnormalities (mainly acinar necrosis and loss of beta-cell staining) in the islets of Langerhans in the treated lambs, but it could not be concluded that auto-immunisation against somatostatin produced any specific morphological change in the betacells of the pancreatic islets.The results of this study suggest that somatostatin plays an important physiological role in regulating growth stimulation. Removal of the inhibitory effect of somatostatin (in this case by neutralisation of the effect with antibodies) can result in increased growth. The hormonal changes induced by this treatment have yet to be fully elucidated. Further investigations are required to assess the usefulness of this treatment as an alternative to the more conventional synthetic steroid treatments for growth promotion.     

Effects of intramuscular meloxicam administration on prostaglandin E2 synthesis in the North American bullfrog (Rana catesbeiana)

Meloxicam is a commonly used nonsteroidal anti-inflammatory drug (NSAID) in veterinary medicine, but its use in amphibians has not been reported in the literature. NSAIDs are known to act by providing anti-inflammatory and analgesic actions by inhibiting the synthesis of prostaglandin E2 (PGE2). The objective of this study was to evaluate whether the intramuscular administration of meloxicam would decrease the circulating serum PGE2 levels in the North American bullfrog (Rana catesbeiana) following tissue trauma induced by a punch biopsy. Eighteen adult North American bullfrogs were randomly assigned to two treatment groups: meloxicam (0.1 mg/kg i.m.) and control (0.9% saline i.m.). Blood was obtained via cardiocentesis immediately prior to administration of the two treatment regimes and serum was frozen. A 4-mm punch biopsy was taken from the right triceps femoris muscle to induce an inflammatory response. Twenty-four hours later, a second blood sample was collected and serum was harvested and frozen. Serum PGE2 concentrations were measured using a commercial PGE2 enzyme assay (EIA) kit. Twenty-four hours following the biopsy, the mean circulating PGE2 levels of animals treated with meloxicam was 57.79 +/- 12.35 pg/ml, which did not differ significantly from animals that were treated with saline (85.63 +/- 17.55 pg/ml, P > or = 0.05). The calculated means of the absolute change between the circulating baseline PGE2 levels and the postinjury circulating PGE2 levels were significantly lower in animals treated with meloxicam (13.11 +/- 17.31 pg/ml) than in control animals treated with saline (46.14 +/- 38.02 pg/ml) (P < or = 0.05). These results suggest that the systemic administration of meloxicam at a dosage of 0.1 mg/kg once daily suppresses circulating serum PGE2 levels postinjury in the North American bullfrog.     

Genetic variations in the antibody response in young bulls

Considerable evidence for the existence of a direct genetic control of the immune response has been presented during recent years. Experimental work with rodents are the main basis for this evidence. The first study on genetic variations in the antibody response was carried out by Gorer & Schütze (1938). Later Cinader (1960) published detailed considerations about the specificity and inheritance of the antibody response. In mice it has been demonstrated that a few dominant immune response (Ir) genes determine the ability to produce antibodies against certain specific antigens (McDevitt & Tyan 1968). The magnitude of the response is probably under the influence of polygenes, which are not associated with Ir genes. This theory is supported by selection for high and low antibody production in mice (Biozzi et al. 1972).