Adaptation of IFN-gamma ELISA and ELISPOT tests for feline tuberculosis

There are currently no reliable immunodiagnostic tests for feline tuberculosis. Infection of domestic cats in the UK is thought to occur via their contact with the relevant reservoir of infection, e.g. cattle and badgers for Mycobacterium bovis, and rodents for M. microti. In the African National Parks, where M. bovis infection of Bovidae is an increasing problem, transmission to big cats is occurring via their ingestion of infected carcasses. We have adapted feline ELISA and ELISPOT assays to potentially provide the first cell-based diagnostic test for the detection of tuberculosis in cats. We tested peripheral blood mononuclear cell antigen-specific IFN-gamma responses of 18 cats suspected of mycobacterial infection for which biopsy material was co-submitted to the Veterinary Laboratories Agency for mycobacterial culture and identification. Seventeen cats were tested by ELISA while seven cats were tested by ELISPOT (six cats were tested by both ELISA and ELISPOT). Six healthy control cats provided baseline data for these tests. Responses to bovine and avian tuberculins (PPDB and PPDA) and a protein cocktail of ESAT6 and CFP10 were measured, together with positive mitogen (PMA and calcium ionophore) and negative (medium) controls. Overall, both ELISPOT and ELISA tests were found to be suitable for generating rapid results (2 and 4 days, respectively), which provided good predictive information for M. bovis and M. microti infections, but were unable to reliably discern M. avium infection.     

Characterization of specific antibodies and the establishment of sandwich ELISA and ELISPOT systems for swine IL-4

We produced four monoclonal antibodies (mAb) and two polyclonal antibodies using the purified cytokine expressed in bacteria and characterized them. Specific binding of each of the mAb and polyclonal antibodies to recombinant swine IL-4 (rSwIL-4) purified from Escherichia coli and baculovirus was demonstrated in an indirect ELISA and/or in western blotting. We established a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring concentration of SwIL-4 in biological samples and established an enzyme-linked immunospot (ELISPOT) assay for detecting IL-4-secreting cells using a mAb and a polyclonal IgG from goat. The detection limit of the sandwich ELISA for SwIL-4 was 78 pg/ml. Using sandwich ELISA, SwIL-4 was detected in the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Mycoplasma hyopneumoniae and could quantitate in supernatants of mitogen-stimulated PBMC culture. The ELISPOT system is useful for the detection of IL-4 producing cells in swine PBMC culture.     

IL-4, IL-5 and IFN-gamma mRNA expression in pulmonary lymphocytes in equine heaves

Heaves is a common condition of horses of cold climate that is characterized by small airway inflammation and obstruction following exposure of susceptible horses to moldy hay and straw. It has been shown that helper T lymphocytes (Th) orchestrate the inflammatory response in asthma and in various animal models of allergic lung diseases by the release of Th2-type cytokines. Results of previous studies indicate that a predominant expression of Th2-type response by airway cells may also be present in heaves. To evaluate the temporal mRNA expression of Th1 (IFN-gamma) and Th2 (IL-4, IL-5) type cytokines in heaves and their relationship to clinical disease, we studied the pulmonary mechanics and cytokine mRNA expression (IL-4, IL-5 and IFN-gamma) in bronchoalveolar lavage lymphocytes of horses with heaves (n=6) and control (n=6) before and after 24h and 9 days of continuous natural inhalation challenge. Starting 24h after challenge horses with heaves, but not control horses, had a significant increase in pulmonary elastance and the number of lymphocytes expressing mRNA for IL-4 and IL-5. These changes were further increased at 9 days, at which time the number of cells positive for IFN-gamma mRNA was decreased. In this study we have shown that BAL lymphocytes of horses with heaves during clinical exacerbation have a predominant Th2-type cytokine response and that this response coincides in time with the presence of airway obstruction.     

Blood concentrations of the cytokines IL-1beta,IL-6, IL-10, TNF-alpha and IFN-gamma during experimentally induced swine dysentery

Background

Knowledge of the cytokine response at infection with Brachyspira hyodysenteriae can help understanding disease mechanisme involved during swine dysentery. Since this knowledge is still limited the aim of the present study was to induce dysentery experimentally in pigs and to monitor the development of important immunoregulatory cytokines in blood collected at various stages of the disease.

Methods

Ten conventional pigs (~23 kg) were orally inoculated with Brachyspira hyodysenteriae B204^T. Eight animals developed muco-haemorrhagic diarrhoea with impaired general body condition. Blood was sampled before inoculation and repeatedly during acute dysentery and recovery periods and cytokine levels of IL-1β, IL-6, Il-10, TNF-α and IFN-γ were measured by ELISA.

Results

IL-1β was increased at the beginning of the dysentery period and coincided with the appearance of Serum amyloid A and clinical signs of disease. TNF-α increased in all animals after inoculation, with a peak during dysentery, and IL-6 was found in 3 animals during dysentery and in the 2 animals that did not develop clinical signs of disease. IL-10 was found in all sick animals during the recovery period. IFN-γ was not detected on any occasion.

Conclusion

B. hyodysenteriae inoculation induced production of systemic levels of IL-1β during the dysentery period and increased levels of IL-10 coincided with recovery from dysentery.     

Development of an ELISA for bovine IL-10

The objective of the study was to develop an assay for bovine IL-10 that could be applied to analyses of immune responses and advance understanding of a variety of diseases of cattle. Recombinant bovine IL-10 (rbo IL-10) was transiently expressed in Cos-7 cells and shown to inhibit the synthesis of IFN gamma by bovine cells stimulated with antigen in vitro. Mice were immunised with a plasmid containing a cDNA insert encoding rbo IL-10 and inoculated with rbo IL-10. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-10 in an ELISA. Some of these mAb neutralised the ability of rbo IL-10 to inhibit IFN gamma synthesis by antigen-stimulated bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-10 present in supernatant of PBMC stimulated with ConA. A luminescent detection method was applied to the ELISA making it more sensitive. Using this method native IL-10 was detected in supernatants of PBMC, diluted blood and undiluted blood from cattle immunised with Mycobacterium bovis BCG or ovalbumin and incubated in vitro with antigen indicating the applicability of the assay to a number of in vitro culture systems.     

Cell-mediated immune responses to a killed Salmonella enteritidis vaccine: lymphocyte proliferation, T-cell changes and interleukin-6 (IL-6), IL-1, IL-2, and IFN-gamma production

Two experimental approaches were used to investigate the immunological responses of chickens to a commercial killed Salmonella enteritidis (SE) vaccine. In the first, the effects of host age on antigen-specific proliferative responses and cytokine production were examined. Compared with non-vaccinated controls, 4-wk-old vaccinated chickens showed higher proliferation to SE LPS and flagella. The lymphoproliferation responses to these antigens of 8-mo-old vaccinated chickens were not different compared to the non-vaccinated controls. Increased production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) by antigen-stimulated splenocytes following vaccination were, in general, more often observed in 4-wk-old compared with 8-mo-old chickens, whereas serum levels of these cytokines were consistently higher in the vaccinated birds compared with controls regardless of age. The second set of experiments were designed to determine the effects of SE vaccination on mitogen- or antigen-induced splenocyte proliferation and serum nitric oxide (NO) and cytokine levels. Splenocytes from vaccinated chickens stimulated with SE flagella showed significantly increased numbers of TCRgammadelta+ cells at 7 days post-vaccination compared with non-vaccinated birds. In contrast, no differences were noted with CD4+, CD8+, or TCRalphabeta+ cells at any time points examined. Higher levels of NO production were observed following stimulation with SE flagella at 4, 7, 11, and 14 days after SE vaccination while serum levels of IFN-gamma, IL-1, IL-6, and IL-8 were elevated only at day 7 post-vaccination. In conclusion, younger chickens mounted a more robust antigen-specific immune response to the SE vaccine compared with older birds and vaccination induced not only T-cell-mediated responses but also host innate and pro-inflammatory responses.     

Cyclosporine A inhibits the mRNA expressions of IL-2, IL-4 and IFN-gamma, but not TNF-alpha, in canine mononuclear cells

The effects of the calcineurin inhibitors cyclosporine A (CsA) and FK506 on the mRNA expressions of various cytokines were evaluated in dogs to determine whether the effects of CsA and FK506 in dogs were similar to those in humans. The mRNA expression levels of the cytokines IL-2, IL-4, IFN-gamma and TNF-alpha were measured in PHA-stimulated canine PBMC using real-time RT-PCR after incubation with CsA or FK506 for 5 hr. Both reagents inhibited IL-2, IL-4 and IFN-gamma mRNA expressions in a dose-dependent manner. However, CsA hardly inhibited the mRNA expression of TNF-alpha. These findings are important for assessing the indications of CsA treatment in dogs.     

Histamine bronchoprovocation does not affect bronchoalveolar lavage fluid cytology, gene expression and protein concentrations of IL-4, IL-8 and IFN-gamma

In diagnosing inflammatory airway disease (IAD) in performance horses, a histamine bronchoprovocation (HBP) test is often performed. In previously published studies, HBP is usually undertaken prior to cytological examination of the bronchoalveolar lavage (BAL) cells. The purpose of this study was to determine if HBP alters (1) the total nucleated cell numbers and distribution in BAL fluid (BALF) and (2) the mRNA and protein concentrations of selected cytokines in BAL cells and BALF, respectively. BALF was initially collected endoscopically from the right middle or diaphragmatic lung lobe in eight healthy young Standardbred horses. Five to six days later, HBP was performed by aerosolization of histamine (8mg) over a 2min period. BALF was again collected within 2-4h of the HBP from the left middle or diaphragmatic lung lobe. In both samples, total and differential WBC counts were obtained. The gene expressions of interleukin-4 (IL-4), IL-8, interferon-gamma (IFN-gamma) and beta-actin in BAL cells were measured using real-time RT-PCR. The cytokine protein concentrations were measured in the BALF using ELISA. HBP was not associated with either a change in the total BAL cell number or in the distribution of the BAL cells. BAL cell expression of IL-4, IL-8 and IFN-gamma, detected in all samples with the exception of IL-4 in one horse (post-HBP), was not altered as a result of HBP. HBP was not associated with a significant change in IL-8 or IFN-gamma concentrations in the BALF. IL-4 protein was undetectable in BALF either prior to or following HBP. We conclude that HBP can precede BALF collection performed within 2-4h of the former without affecting selected parameters analysed in the BAL cells or BALF.     

Bacterial lipopolysaccharide stimulates bovine neutrophil production of TNF-alpha, IL-1beta, IL-12 and IFN-gamma

After intramammary infection, polymorphonuclear neutrophil leukocytes (PMN) are the first cells recruited into the mammary gland. Rapid recruitment of and bacterial phagocytosis and killing by PMN are the most effective defenses against establishment of bacterial infection. In addition to their phagocytic and bactericidal properties, PMN may play a key supportive role through secretion of cytokines during the innate immune response. We sought to determine whether bovine PMN produce cytokines in response to stimulation by lipopolysaccharide (LPS). To investigate the effects of LPS on the expression of cytokines secreted by bovine PMN, we measured the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-12, and interferon (IFN)-gamma by ELISA after stimulation with different concentrations of LPS, and secretion of IL-8 after co-stimulation with LPS and either TNF-alpha or IL-1beta. Bovine PMN were shown to secrete TNF-alpha , IL-1beta, IL-12, IL-8 and IFN-gamma in response to LPS. Co-incubation of PMN with LPS and TNF-alpha increased secretion of IL-8 when compared to LPS alone. It was concluded that LPS stimulation up-regulates the secretion of cytokines by bovine PMN, and that co-incubation of LPS with TNF-alpha had an additive effect on the secretion of IL-8. These data show that bovine PMN, in addition to their phagocytic and bactericidal properties, may play a supportive role in the innate immune response to infection by Gram-negative bacteria through their ability to produce immuno-regulating cytokines.     

Mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus

Eleven-day-old conventionally reared piglets were inoculated orally with two different doses of the cell-culture adapted strain CV-777 of the porcine epidemic diarrhoea virus (PEDV) or the virulent isolate of the same strain and challenged with the same virulent PEDV 3 weeks later. Pigs inoculated with the two doses of the attenuated virus did not show any typical sign of the disease, and virus shedding was not frequent. In contrast, 31% of pigs exposed to the virulent PEDV developed diarrhoea and virus shedding was demonstrated in 100%. At different postinoculation day (PID) and postchallenge day (PCD) virus-specific antibody-secreting cells (ASC) in gut associated lymphoid tissues (duodenum and ileum lamina propria and mesenteric lymph nodes) and systemic locations (blood and spleen) were assessed by enzyme-linked immunospot (ELISPOT). Only a small response was detected in the groups inoculated with attenuated PEDV, whereas in the group previously exposed to the virulent virus on PID 21 a large number of IgG and IgA ASC was detected. Isotype-specific antibody responses in serum were investigated by ELISA. IgG responses were detected in all groups, although the highest response corresponded to the group inoculated with virulent virus and only this group showed an IgA response. The pigs exposed to virulent PEDV were completely protected against the challenge with a higher dose of the same virulent virus on PID 21 and none of them shed the virus. The pigs inoculated with the attenuated strain were partially protected against the challenge, and 25% of the low dose- and 50% of the high dose-exposed pigs did not shed virus after challenge. All the pigs from a control group, not previously exposed to the virus, excreted the virus in faeces. A strong positive correlation was established between protection and the ASC responses detected in gut associated lymphoid tissues and blood at the challenge day and also between protection and serum isotype-specific antibody titers on that day. In addition, the IgA and IgG ASC responses detected in the blood on PID 21 also correlated with the responses found in the gut associated lymphoid tissues. The ASC and serum antibody responses after the challenge corresponded to a secondary immune response in the groups inoculated with attenuated virus, whereas a primary response was evident in the control group. No increase was seen in any of the parameters studied in the pigs inoculated with virulent PEDV.     

Pituitary and adrenocortical responses to corticotropin-releasing factor in pigs

A study was conducted to determine whether differences in adrenocortical response to exogenous adrenocorticotrophin (ACTH) were an accurate reflection of an animal's perception of and response to stressful stimuli, or whether the pituitary gland might modulate adrenocortical responsiveness. Sixteen Large White x Landrace female pigs, of which 8 had high adrenocortical response to ACTH and the other 8 had low response, were administered IV a bolus of synthetic human corticotropin-releasing factor (hCRF) at dose rates ranging from 0.002 to 2 micrograms/kg of body weight. Blood samples were collected at known times for up to 2 hours after administration of hCRF. Plasma ACTH and cortisol concentrations were measured by radioimmunoassay. Results indicate that hCRF stimulated the pituitary gland of high- and low-responding pigs to secrete ACTH, which in turn stimulated the adrenal cortex to secrete cortisol. Plasma ACTH concentration, before or after hCRF administration, was not significantly different between the high and low responders. However, high-responding pigs had higher cortisol concentration after hCRF administration than did low-responding pigs. Thus, the differences in adrenocortical response to ACTH between the 2 groups of pigs were not attenuated by variation in pituitary response. It is concluded that adrenocortical responsiveness to ACTH is an accurate indicator of the perception of and the response to stress.     

Use of immunoenzyme (ELISA) diagnosis of Aujeszky's disease in pigs during the recovery period

Samples of blood and blood serums of pigs were examined for the presence of antibodies to the Aujeszky's disease virus. The virus-neutralizing (VN) test and the enzymoimmunologic (ELISA) method were used for this examination. As indicated by comparison of the average titres of antiviral antibodies determined by both methods, the ELISA method is 60 to 600 times more sensitive than the VN test. The high sensitivity of the ELISA method enabled to detect antiviral antibodies even in samples considered as negative after VN-testing. The method has been used with success for the sanitation of three swine stocks where the Aujeszky's disease was eradicated without interruption of operation.     

Synergistic effects of IL-2, IL-12 and IL-18 on cytolytic activity, perforin expression and IFN-gamma production of porcine natural killer cells

Natural killer (NK) cells are one of the main cellular components of the innate immune system. They play an important role in the immune response against infections as well as tumour cells and therefore have two major properties: production of immune regulatory cytokines and chemokines as well as cytolytic destruction of particular target cells. The existence of NK cells in swine is well known as well as the phenotype of resting NK cells, but their response following activation by cytokines is still poorly understood. Therefore, we tested the influence of the immune regulatory cytokines IL-2, IL-12 and IL-18 on cytolytic activity, phenotype, IFN-gamma production and the accumulation of perforin in cytoplasm of peripheral blood mononuclear cells (PBMC) as well as purified NK cells. NK cells were enriched from PBMC using a magnetic cell separation (MACS) strategy with monoclonal antibodies against CD3, CD21 and SWC3, thereby removing T-, B- and myeloid cells. Respective fractions were used in flow cytometry (FCM) based cytolytic assays with the human tumour cell line K562 as target. After stimulation with the cytokines described above, the NK cell enriched CD3(-)CD21(-)SWC3(-) fraction showed an evident increase in the cytolytic activity compared to PBMC. This enhanced cytolytic activity was accompanied by a strong enrichment of IFN-gamma producing cells when a combination of all three cytokines (IL-2/IL-12/IL-18) was used; as determined in ELISPOT assays and intracellular staining of IFN-gamma in FCM. Also, the combination of these three cytokines led to an accumulation of perforin in the cytoplasm and an up-regulation of CD25 compared to control cultures incubated in medium without cytokines. The experiments performed clearly indicate a stimulatory role and strong synergistic effects of the investigated cytokines in the activation of porcine NK cells in vitro, inducing IFN-gamma, perforin production and cytotoxicity against target cells.     

Use of ELISA to assess lungworm infection in calves

The enzyme-linked immunosorbent assay (ELISA) technique was applied to the detection of lungworm infections in calves. In experimentally infected animals different responses to larval and adult worm antigens were observed. The response to adult worm antigens was delayed in vaccinated animals when infection occurred by the gradual uptake of infective larvae from contaminated pasture. A serological survey in The Netherlands demonstrated a high incidence of lungworm infection in both vaccinated and unvaccinated herds. There was a good correlation between anti-adult worm and anti-larval ELISA-titres. ELISA appeared to be a useful technique for assessing the level of lungworm infection in a herd.     

Integrated adrenal, somatotropic, and immune responses of growing pigs to treatment with lipopolysaccharide

The objective of this research was to provide an integrated look at systemic adrenal, somatotropic, and immune responses of growing pigs to challenge with lipopolysaccharide (LPS). Weaned pigs were challenged intraperitoneally with 100 microg/kg BW of LPS or sterile saline, and rectal temperature and blood data were collected for 72 h. Daily feed intake also was monitored. Plasma was analyzed for concentrations of cortisol, tumor necrosis factor alpha (TNFalpha), the acute phase protein haptoglobin, growth hormone (GH), insulin-like growth factor I (IGF-I), and prostaglandin E2 (PGE2). As expected, LPS decreased feed intake, stimulated a febrile response, and activated the hypothalamic-pituitary-adrenal (HPA) axis as demonstrated by increased cortisol levels. Cortisol reached maximum elevation 2 h after treatment (P < .001) and remained elevated through 12 h (P < .001). Circulating TNFalpha was increased by LPS at 2 and 4 h after treatment (P < .001), and an apparent (not statistically significant) increase in haptoglobin also occurred in challenged animals. The LPS injection suppressed IGF-I by 2 h following treatment (P < .01), and circulating IGF-I remained reduced relative to controls through 44 h. Overall, GH was increased in LPS-treated pigs (P < .05), although the treatment x time interaction was not significant. Plasma PGE2 was increased transiently at 2 h (P < .05) and then subsequently suppressed at 4, 8, and 12 h following LPS (P < .05). This study provides a comprehensive view of systemic effects of LPS on components of the HPA, growth, and immune axes. In addition, these are the first data to document changes in circulating PGE2 in unrestrained animals during the early hours of the acute phase response to LPS.     

Assessment of T-dependent and T-independent immune responses in cattle using a B cell ELISPOT assay

Understanding the mechanisms that maintain protective antibody levels after immunisation is important for vaccine design. In this study, we have determined the kinetics of plasma and memory B cells detectable in the blood of cattle immunised with model T-dependent or T-independent antigens. Immunisation with the T-D antigen resulted in an expansion of TNP-specific plasma cells post-TNP primary and booster immunisations, which was associated with increased titres of TNP-specific IgG antibodies. Although no TNP-specific memory B cells were detected in the T-D group following the primary immunisation, we detected an increase in the number of TNP-specific memory B cells post-TNP boost. In contrast, no TNP-specific plasma or memory B cells were detected after primary or secondary immunisation with the T-I antigen. We then investigated if immunisation with a third party antigen (tetanus toxin fragment C, TTC) would result in a bystander stimulation and increase the number of TNP-specific plasma and memory B cells in the T-D and/or T-I group. TTC immunisation in the T-D group resulted in a small increase in the number of TNP-specific plasma cells post-TTC primary immunisation and boost, and in an increase in the number of TNP-specific memory B cells post-TTC boost. This bystander effect was not observed in the animals previously immunised with the T-I antigen. In conclusion, the present study characterised for the first time the B cell response in cattle to immunisation with T-D and T-I antigens and showed that bystander stimulation of an established T-D B cell memory response may occur in cattle.     

Motor responses to stimulation during isoflurane anaesthesia in pigs

Objective To investigate motor responses to stimulation during the transition from ‘deep’ (burst suppression) to ‘light’ isoflurane anaesthesia in pigs. Study design Prospective, randomized observational study. Animals Five castrated male and five female Norwegian landrace pigs, weighing 19–29 kg. Materials and methods Anaesthesia was induced with isoflurane and the inspired concentration gradually increased until a burst suppression electroencephalogram (EEG) was recorded. End‐tidal isoflurane concentration ( F ) was then allowed to equilibrate for 30 minutes after which the eyelashes, cornea, nasal septum, anus, interdigital skin fold, periople, tail and claw were stimulated. The motor response to stimulation at each location was graded from 0 to 5. End‐tidal isoflurane concentration was decreased 0.3% and the areas re‐stimulated; this was repeated three times in each pig. A linear regression analysis using response as dependent and anaesthetic level as independent variable was performed for each stimulus in each pig. Using Student's t‐statistic a 95% confidence interval for the mean slope of each stimulus was constructed. Results No pig responded to eyelash brushing. The mean slopes for the other stimuli indicated increasing responses with decreasing F . Responses to periople pinching and tail and claw clamping showed significant increases. No stimuli consistently increased the magnitude of response in all pigs, and the appearance and absence of a response was inconsistent between pigs. Motor responses occurred in at least one pig during isoflurane burst suppression anaesthesia to all stimuli except eyelash brushing. Conclusions All the stimuli investigated may elicit movement responses during burst suppression anaesthesia with isoflurane except eyelash brushing. No consistent response pattern between pigs was observed with decreasing isoflurane concentration. Of the stimuli evaluated, clamping the tail or claw and pinching the periople appear the most reliable indicators of anaesthetic depth. Clinical relevance The absence or presence of single reflexes does not accurately reflect the degree of isoflurane‐induced cortical depression in individual pigs.     

Isotype-specific antibody-secreting cells in systemic and mucosal associated lymphoid tissues and antibody responses in serum of conventional pigs inoculated with PEDV

An enzyme-linked immunospot (ELISPOT) has been developed to detect porcine epidemic diarrhea virus (PEDV)-specific antibody secreting cells (ASC) in gut associated lymphoid tissues (duodenum and ileum lamina propria and mesenteric lymph nodes) and systemic locations (spleen and blood) of conventional pigs so as to characterise the mucosal and systemic antibody response generated by the infection with PEDV. A total number of 28 eleven-day-old conventional pigs were orally inoculated with the field isolate of the PEDV strain CV-777. Diarrhea was observed in 32% of the pigs and virus shedding was demonstrated in 100% between postinoculation day (PID) 1 and 8. Serum IgG and IgA antibodies to PEDV were detected by isotype ELISA from PID 12 and 15, respectively, reaching maximum values at PID 32 (IgG) and 21 (IgA). PEDV specific IgM ASC occurred in all the tissues between PID 4 and 7, with the strongest response in the intestinal lamina propria. IgA and IgG ASC responses were evident in the intestinal lymphoid tissues from PID 21, the highest number of specific ASC corresponded to the duodenum lamina propria. In the systemic lymphoid tissues the number of IgG and IgA ASC detected were lower than in the mucosal tissues, however, in the blood, presence of IgA ASC was constantly detected from PID 14 until the end of the experiment. Memory antibody response to the PEDV was also studied by secondary in vitro stimulation of the mononuclear cells (MNC) isolated from mesenteric lymph nodes, spleen and blood. The memory B cell response was prominent at PID 21 and 25 and consisted in IgG and IgA ASC. To our knowledge, this is the first report to research into the presence and distribution of specific ASC in different locations of the systemic and the gut associated lymphoid tissues after a PEDV infection as well as the presence of memory B cells.     

Use of innate behaviors to evaluate sensory function in the dog

The need to evaluate sensory function in the dog is discussed. A group of techniques that use innate behaviors (species-typical behaviors) and are effective in the evaluation of sensory function of domestic animals are described. Techniques to measure olfactory, gustatory, auditory, and visual function are included.