Identification and genotyping of feline infectious peritonitis-associated single nucleotide polymorphisms in the feline interferon-γ gene

Feline infectious peritonitis (FIP) is an immune-mediated, highly lethal disease caused by feline coronavirus (FCoV) infection. Currently, no protective vaccine or effective treatment for the disease is available. Studies have found that some cats survive the challenge of virulent FCoV isolates. Since cellular immunity is thought to be critical in preventing FIP and because diseased cats often show a significant decrease in interferon-γ (IFN-γ) production, we investigated whether single nucleotide polymorphisms (SNP) in the feline IFN-γ gene (fIFNG) are associated with the outcome of infection. A total of 82 asymptomatic and 63 FIP cats were analyzed, and 16 SNP were identified in intron 1 of fIFNG. Among these SNP, the fFING + 428 T allele was shown to be a FIP-resistant allele (p = 0.03), and the heterozygous genotypes 01C/T and +408C/T were found to be FIP-susceptible factors (p = 0.004). Furthermore, an fIFNG + 428 resistant allele also showed a clear correlation with the plasma level of IFN-γ in FIP cats. For the identification of these three FIP-related SNP, genotyping methods were established using amplification refractory mutation system PCR (ARMS-PCR) and restriction fragment length polymorphisms (RFLP), and the different genotypes could easily be identified without sequencing. The identification of additional FIP-related SNP will allow the selection of resistant cats and decrease the morbidity of the cat population to FIP.     

Phagolysosome maturation of macrophages was reduced by PE_PGRS 62 protein expressing in Mycobacterium smegmatis and induced in IFN-γ priming

Mycobacterium bovis parasitizes host macrophages and has developed strategies to survive within macrophages. Research on mycobacteria-specific PE_PGRS genes indicates that they code for cell surface proteins that may influence virulence. To further elucidate the molecular pathogenesis of tuberculosis and host response to M. bovis, we explored the mechanisms by which PE_PGRS62 protein increase persistence of mycobacterium within host macrophages. We found that the M. smegmatis strain expressing M. bovis PE_PGRS 62 protein reduced phagolysosome maturation in human macrophages, and significantly decreased the mRNA expression of IL-1β in a dose- and time-dependent. We identified that IFN-γ priming of macrophages immediately prior to infection with PE_PGRS62 expressing M. smegmantis, enhanced the maturation of phagolysosomes and induced IL-1β production both that the protein and mRNA levels and further activated the NF-κB pathway. Overall, we demonstrated that PE_PGRS62 protein altered the immune environment of the host cells, which suggested that the pathogenic PE_PGRS62 protein altering the immune mechanism might be involved in the pathogenesis of mycobacterial disease and hence influenced host cell responses to M. bovis infection.     

Upregulation of TNF-α Production by IFN-γ and LPS in Cultured Canine Keratinocytes: Application to Monosaccharides Effects

Activated keratinocytes play a key role in the cutaneous immune system by their interactions with other cell types through the production of cytokines with both autocrine and paracrine activity. But there is little knowledge about epidermal cytokines in the dog. In this study, cultured canine keratinocytes were stimulated by human recombinant interferon γ (IFN-γ) and lipopolysaccharide (LPS) and cell supernatants were tested for tumour necrosis factor α (TNF-α) concentration using a cell viability assay on a murine cell line. We show that IFN-γ in combination with LPS significantly increases TNF-α secretion by canine keratinocytes. The best stimulations were obtained using confluent cultures and the association of IFN-γ (400 ng/ml) and LPS (40 μg/ml). The experimental protocol we describe represents a new method for studying keratinocyte activation and its modulation in the dog. We provide an example of application of our method: the study of the effects of different monosaccharides on canine keratinocyte activation.     

Does blood contamination of urine compromise interpretation of the urine protein to creatinine ratio in dogs?

AIMS: To determine the effect of contamination of urine with 0–5% blood, varying in haematocrit and protein concentrations, on the urine protein to creatinine ratio (UPC) in dogs, and to determine whether the colour of urine can be used to aid interpretation of UPC results.

METHODS: Urine samples were collected by free catch from 18 dogs, all of which had UPC?<0.2. Venous blood samples were also collected from each dog, and the blood from each dog was added to its own urine to produce serial concentrations of 0.125–5% blood. The colour of each urine sample was recorded by two observers scoring them as either yellow, peach, orange, orange/red or red. Protein and creatinine concentrations were determined, and dipstick analysis and sediment examination was carried out on each sample. Based on colour and dipstick analysis, samples were categorised as either having microscopic, macroscopic or gross haematuria. A linear mixed model was used to examine the effect of blood contamination on UPC.

RESULTS: The uncontaminated urine of all 18 dogs had a UPC?<0.2. Adding blood to the urine samples resulted in an increase in UPC at all contamination concentrations compared to the non-contaminated urine (p<0.001). None of the 54 samples with microscopic haematuria had UPC?>0.5. For 108 samples with macroscopic haematuria the UPC was >0.5 in 21 samples (19.4 (95% CI=13.1–27.9)%), and for 54 samples with gross haematuria 39 (72 (CI=59.1–82.4)%) had a UPC?>0.5. No samples had a UPC?>2.0 unless the blood contamination was 5% and only 3/18 (17%) samples at this blood contamination concentration had a UPC?>2.0.

CONCLUSIONS AND CLINICAL RELEVANCE: This study showed that while blood contamination of ≥0.125% does increase the UPC, if the urine remains yellow (microscopic haematuria), then there is negligible chance that a UPC?>0.5 will be solely due to the added blood. In that scenario, attributing the proteinuria present to the haematuria in the sample would be inappropriate. However blood contamination that results in discolouration of the urine sample from yellow (indicating macroscopic or gross haematuria) could increase the UPC above the abnormal range and would need to be considered as a differential for the proteinuria. Thus knowledge of urine colour, even if limited to simple colour scores (yellow, discoloured, red) could be utilised to aid interpretation of the UPC in samples with haematuria.     

Relationship between contractions of the uterus and concentration of PGF2α in uterine venous blood after luteolysis in gilts

The origin and physiological significance of high pulses of prostaglandin F2α (PGF2α) in uterine venous blood that occur 2-3 days after luteolysis are not well understood. We studied the relationship between contractions of the uterus evoked by exogenous oxytocin (OT) and PGF2α concentration in uterine venous blood on day 17 of the porcine oestrous cycle. The infusion of OT into the uterine artery produced an immediate increase in the uterine intraluminal pressure (UIP) (p < 0.001) and a simultaneous elevation in PGF2α concentration in uterine venous blood (p < 0.0001). The infusion of indomethacin (IND) into the uterine artery slightly decreased PGF2α concentration in uterine venous blood, but it did not suppress uterine contraction or the rapid increase in PGF2α concentration in uterine venous blood just after OT infusion (p < 0.0001), which was lower that in gilts not treated with IND. We conclude that the spikes of PGF2α concentration in uterine venous blood occurring after OT infusion on day 17 of the porcine oestrous cycle are mainly caused by the excretion with venous blood from the remodelled uterus and that PGF2α synthesis may contribute to this. These results suggest that the high spikes in PGF2α concentration that occur 2-3 days after luteolysis in pigs, sheep, cows and mares all have a similar origin.     

Correlation of antigen-specific IFN-γ responses of fresh blood samples from Mycobacterium avium subsp. paratuberculosis infected heifers with responses of day-old samples co-cultured with IL-12 or anti-IL-10 antibodies

Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring cell-mediated immune responses using the interferon gamma (IFN-γ) assay. Whole blood samples are cultured overnight with specific MAP antigens followed by quantification of IFN-γ by ELISA. It is recommended that the time interval from sampling to culture does not exceed eight hours but addition of the co-stimulating cytokine interleukin 12 (IL-12) or anti-IL-10 antibodies to culture have been demonstrated to enhance IFN-γ responses of cultures stimulated with Johnin purified protein derivative (PPDj). Here we examined the correlation of IFN-γ production in response to PPDj and 15 recombinant antigens in day-old blood samples from heifers 10-21 months of age from a MAP infected herd with addition of either recombinant bovine IL-12 or anti-bovine IL-10 antibody with IFN-γ production in sample day samples. IFN-γ responses of sample day samples showed high correlation with responses to some antigens in day-old samples with addition of IL-12 or anti-IL-10 antibodies to cultures, indicating that day-old protocols can be applied as an alternative to the conventional IFN-γ protocol. Immunogenicity of the novel antigens was generally low for day-old samples. The most promising antigen using the day-old protocol with addition of IL-12 was latency protein LATP-2 as correlations, immunogenicity and diagnostic specificity collectively was high. The latency protein LATP-1 was the most promising antigen in the day-old protocol with addition of anti-IL-10 antibodies.     

Biochemical and hematological changes in the blood of horses after the "Velká Pardubická" steeple chase]

Blood parameters were studied in two groups of horses in the "Velká Pardubická" steeple-chase in 1974, 1975 and 1976. After the race, the levels of lactate showed a manifold increase; an increase was also ascertained in the levels of glucose, sodium, potassium, haemoglobin, in the haematocrit value and in the number of erythrocytes. The following parameters significantly dropped: the levels of acid-base balance - pH, base excess, bicarbonate levels. It was proved that the values of the same parameters in horses during training were incomparably lower. It is advisable to examine horses thoroughly during training and to use the results of training for the evaluation of their condition before difficult races.     

Co-incubation with IL-18 potentiates antigen-specific IFN-γ response in a whole-blood stimulation assay for measurement of cell-mediated immune responses in pigs experimentally infected with Lawsonia intracellularis

The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in vitro assay for a direct read-out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult to evaluate for less severe or immunocompromising infections. Here we investigated the performance of the assay when recombinant co-stimulatory cytokines IL-12 and/or IL-18 were added along with Ag or PBS to cultures of whole-blood from pigs infected with Lawsonia intracellularis. In pigs recovering from a natural infection, addition of rIL-12 or rIL-18 alone increased the Ag-specific IFN-γ release while addition of both cytokines resulted in increased IFN-γ release also in PBS cultures. In analyses after experimental infections with L. intracellularis, significant increased levels of Ag-specific IFN-γ production were measured in Ag+rIL-18 cultures from infected pigs compared to the background response in PBS+rIL-18 control samples (p<0.01) or to Ag+rIL-18 cultures from non-inoculated control pigs (p<0.05). Flow cytometry identified two lymphocyte subsets as the Ag-specific IFN-γ producers. The highest IFN-γ production was by CD4(+)CD8(+) cells while a more numerous population of CD4(-)CD8(+) cells produced lower amounts of IFN-γ in response to rIL-18 and L. intracellularis Ag.     

Effects of clopidogrel therapy on whole blood platelet aggregation,the Plateletworks® assay and coagulation parameters in cats with asymptomatic hypertrophic cardiomyopathy: a pilot study

Background: Although scientific evidence is limited, clopidogrel is frequently used as prophylaxis for arterial thromboembolism in cats with hypertrophic cardiomyopathy (HCM).

Objectives: Evaluating effects of clopidogrel therapy in asymptomatic cats with HCM on (1) conventional whole blood aggregation (WBA), (2) alternative platelet aggregation assessed with tubes of the Plateletworks® assay and (3) standard coagulation parameters.

Animals and methods: Prospective, randomized, double-blind, placebo-controlled pilot study. Fourteen asymptomatic HCM cats were randomly allocated to receive placebo (n = 5) or clopidogrel (18.75 mg/cat q24h, n = 9) as part of a larger study. Aggregation responses (to 20 µM adenosine diphosphate (ADP) and 10 µg/ml collagen) in WBA and the Plateletworks® assay and standard coagulation parameters were evaluated at baseline and after seven days of therapy.

Results: Clopidogrel therapy significantly reduced aggregation responses to ADP and collagen in the Plateletworks® agonists tubes (ADP and collagen: P < 0.001), but did not significantly reduce aggregation responses to ADP and collagen in the WBA technique (ADP: P = 0.07, collagen: P = 0.30). Clopidogrel therapy did not show a significant effect on prothrombin time, activated partial thromboplastin time, antithrombin, D-dimers and fibrinogen concentrations.

Conclusion and clinical importance: Clopidogrel therapy at a dose of 18.75 mg/cat q24h for seven days causes a significant decrease in in vitro platelet aggregation evaluated with the Plateletworks® assay, without affecting standard coagulation parameters in cats with asymptomatic HCM.     

IFN-γ expression and infectivity of Toxoplasma infected tissues are associated with an antibody response against GRA7 in experimentally infected pigs

Toxoplasma gondii, an obligate intracellular parasite, can be transmitted to humans via the consumption of infected meat. However, there are currently no veterinary diagnostic tests available for the screening of animals at slaughter. In the current work, we investigated whether cytokine responses in the blood, and antibody responses against recombinant T. gondii GRA1, GRA7, MIC3 proteins and a chimeric antigen EC2 encoding MIC2-MIC3-SAG1, are associated with the infectivity of porcine tissues after experimental infection with T. gondii. Two weeks after experimental infection of conventional 5-week-old seronegative pigs, an IFN-γ response was detected in the blood, with a kinetic profile that followed the magnitude of the GRA7 antibody response. Antibody responses to GRA1, MIC3 and EC2 were very weak or absent up to 6 weeks post infection. Antibodies against GRA7 occurred in all infected animals and were associated with the presence of the parasite in tissues at euthanasia a few months later, as demonstrated by quantitative real-time PCR and isolation by bio-assay. Remarkably, although brain and heart tissue remained infectious, musculus gastrocnemius and musculus longissimus dorsi were found clear of infectious parasites 6 months after experimental infection. Seropositive response in a GRA7 ELISA indicates a Toxoplasma infection in pigs and is predictive of the presence of infectious cysts in pig heart and brain. This new ELISA is a promising tool to study the prevalence of Toxoplasma infection in pigs. Clearance of the infection in certain pig tissues suggests that the risk assessment of pig meat for human health needs further evaluation.     

Effect of β-mannanase on the digestibility of diets with different protein sources in dogs determined by different methodologies

This experiment aimed at evaluating the effects of including the enzyme, β-mannanase, in dog (Canis lupus familiaris) diets based on either poultry (Gallus gallus domesticus) by-product meal (PBM) or soybean [Glycine max (L.) Merr.] Meal (SBM). The second objective was to evaluate 3 methods for determining energy and nutrient digestibility values in diets fed to dogs: total fecal collection (TFC) and use of aia or crude fiber (CF) as a marker. Eight dogs were allotted to a replicated latin square (4 by 4) design. There were 2 diets based on PBM as the major protein source and 2 diets based on SBM as the major protein source. Within each protein source, 1 diet contained no β-mannanase and 1 diet contained 0.01% β-mannanase. Diets were fed for an adaptation period of 5 d followed by 5 d of TFC. Fecal score (1 = watery feces to 5 = dry, hard pellets), pH, DM, and fecal volume were determined. The apparent total tract digestibility (ATTD) of DM, OM, CP, ether extract (EE), N-free extract (NFE), and GE, and ME content were calculated using the methods of TFC, AIA, and CF. Data were analyzed as a 2 by 2 by 3 split-split-plot design (β-mannanase, protein source, and digestibility calculation procedure). There were interactions between protein source and β-mannanase (P < 0.05). Supplementation of β-mannanase increased ATTD of nutrients and energy and ME (+ 195.3 kcal/kg) and also reduced fecal production in the diet with SBM, but not in the diet that contained PBM. There was an interaction between digestibility calculation procedure and protein source (P < 0.05). The use of AIA overestimated ATTD of the diets containing PBM, but digestibility values estimated based on TFC and CF were not different. Dogs fed diets containing SBM produced more feces with greater moisture content and lower pH compared with dogs fed the PBM diet (P < 0.05). Addition of 0.01% β-mannanase increased (P < 0.05) the digestibility and ME content of the diets containing SBM, but did not improve (P > 0.05) fecal texture. Results indicated that values for ATTD of energy and nutrients in diets containing sbm are not different if they are calculated based on TFC, AIA, or CF, but use of AIA may result in an overestimation of values for ATTD of energy and nutrients in diets containing PBM.     

Determination of decisional cut-off values for the optimal diagnosis of bovine tuberculosis with a modified IFNγ assay (Bovigam®) in a low prevalence area in France

The Bovigam(?) gamma interferon (IFNγ) assay was used to complement official skin-test screening in a low bovine tuberculosis (bTB) prevalence region in France. The aim of our work was to determine decisional cut-off values for protein purified derivatives (PPD) and ESAT6-CFP10 antigens (R) in order to optimize the efficacy of the modified Bovigam(?) test, in this low-prevalence area, for optimal classification of infected or non-infected herds following positive skin tests. The sensitivity of the IFNγ assay relative to post-mortem bTB-positive animals (Se(r)) was studied in 60 cattle from 20 bTB-infected herds. Its absolute specificity (Sp) was studied in 492 cattle from 25 bTB-free herds from a bTB-free zone. Its operational specificity (relative to the positive skin test) (Sp(r)) was also studied in 547 skin-test positive cattle from 172 bTB-free herds from an infected zone. Using normalized interpretations for individual (PPD or R) results, the cut-off values at 0.02 for PPD and 0.01 for R were obtained with a view to employ them in low prevalence areas with no previously observed non-specific reactions to SITT. Concerning its use after positive skin tests, cut-off values were set at 0.05 for PPD and at 0.03 for R. The choice of an interpretation method considering positive results with PPD and/or R (PPDUR), justified in a high risk context, provided a test Se(r) of 93% [84-98] and Sp(r) of 71.8% [67.9-75.6]. Analysis of positive results with PPD and R (PPDUR), ideal for low-risk contexts, provided a test Sp(r) of 94.3% [92.0-96.1] and Se(r) of 77% [64-87]. Thus, adapting the criteria to the region's infection status and to the conditions for its application is essential for the appropriate use of the IFNγ assay.     

A live attenuated mutant of Salmonella Montevideo triggers IL-6, IFN-γ and IL-12 cytokines that co-related with humoral and cellular immune responses required for reduction of challenge bacterial load in experimental chickens

A live attenuated Salmonella enterica serovar Montevideo (SM) mutant JOL1599 was constructed by deletion of virulence-associated genes. The protective efficacy and immune response profiles of chickens immunized with JOL1599 were investigated. Immunized chickens demonstrated significant increases in plasma IgG and lymphocyte proliferative responses (P ≤ 0.05). Increased levels of IL-6, INF-γ, and IL-12 were also observed. Immunized birds strongly responded to infection by rapid stimulation of a CD4+ subset of T cells. Organ bacterial recovery assay revealed a significant reduction in the challenge strain among immunized birds. Multiple doses of JOL1599 enhanced the immune responses of the birds as revealed by ascending trends of the immunological profiles. These findings indicate that immunization of chickens with JOL1599 may provide protection against Salmonella Montevideo infection via induction of IL-6, INF-γ, and IL-12 protective cytokines, which in turn triggers conducive humoral and cell-mediated immune responses.     

The effect of a homologous bacterin given to sows prefarrowing on the development of Glässer's disease in postweaning pigs after i.v. challenge with Haemophilus parasuis serotype 5

The trial was carried out to evaluate the impact of maternal antibodies on the development of Gl?sser's disease after i.v. exposure of weaned pigs with a homologous serovar of Haemophilus parasuis (HPS). Two groups of weaned pigs were formed. Group one VI (n = 10): born to vaccinated sows, weaners i.v. challenged one week postweaning and euthanatized 14 days postweaning. Group two NVI (n = 10 wearners): born to non vaccinated sows, i.v. challenged one week postweaning euthanatized 14 days postweaning. One week postweaning all weaners were i.v. inoculated with HPS serovar 5. The following parameters were evaluated: clinical signs (depression, centralnervous signs, fever, lameness), macroscopic lung, pleura, peritoneum, liver and joint changes, and mortality. All trial sows were HPS seronegative prior to vaccination. The HPS vaccinated sows were proven seropositive on day 3 p.p. (values > 0.24), the non vaccinated ones were tested seronegative (values < 0.23). The progeny of sows vaccinated prefarrowing with two doses of HPS serovar 5 bacterin were partially protected against HPS caused clinical and pathological signs. The majority of clinical signs as fever, depression, recumbency, lack of response to verbal stimuli and lameness showed significant (P < 0.05) milder clinical symptoms in VI than in NVI animals. Respiratory signs (P = .169) and involvement of the central nervous system as ataxia, muscular tremor, incoordination of hind legs and convulsions (P = 1) showed no significant differences between the groups. Except lesions of pericard (VI vs. NVI, P = .14) and pleura (VI vs. NVI, P = .14) there were significant (P < 0.05) macroscopic differences at necropsy in lung, liver, joints and cerebrospinal fluid between the offspring of vaccinated sows and the ones of non vaccinated dams. No HPS were isolated from the nasal mucosa of the pigs prior to inoculation. HPS serovar 5 was recovered at necropsy from the nasal mucosa of all pigs in both groups. One pig from group VI presented in all examined organs the presence of HPS serovar 5. The remaining animals in group VI revealed in lung, pericard, pleura, liver, joints and cerebrospinal fluid no presence of HPS. The rate of isolation between VI and NVI groups revealed a significant (P < 0.05) difference. All the survived piglets of group NVI showed positive ELISA titres against HPS serovar 5 (values > .24). The piglets that died or were euthanatized before the end of the study have not been subjected to ELISA serological testing. One piglet died in group VI before the end of the study. Non of the remaining animals in group VI showed seroconversion to HPS serovar 5. Implications: Vaccination of sows did not influence the colonisation of nasal mucosa, but progeny of sows vaccinated prefarrowing with two doses of HPS serovar 5 bacterin were partially protected against HPS caused diseases.     

Production,chemical composition,and fatty acid profile of milk from buffaloes fed with cupuaçu (Theobroma grandiflorum) cake and murumuru (Astrocaryum murumuru) cake in the Eastern Amazon

The objective was to evaluate the effect of concentrate supplementation using by-products of the Amazonian industry on milk production, milk composition, and milk fatty acid profile of dairy buffaloes. Twelve lactating buffaloes (544.5 ± 35.6 kg, 6.4 ± 2.2 years old, 59 ± 6 days in milk) were allotted in a pasture of Mombaça grass and managed under rotational grazing (4 days occupancy/28 days rest). A 3 × 3 Latin square was adopted, and each animal alternately received three supplementary treatments based on corn bran + soybean meal or cupuaçu cake or murumuru cake for 21 days per treatment. Murumuru cake increased the levels of lauric acid and myristic acid in the milk (p < 0.05). Murumuru cake reduced the unsaturated fatty acid contents in the milk compared with animals fed control diet or cupuaçu cake (24.27% vs. 25.24% vs. 25.08%). The n-6/n-3 ratio was 2.6, 1.97, and 2.0 in the control, cupuaçu, and murumuru groups, respectively. Based on this parameter, cakes made from cupuaçu as well as murumuru could be considered to be adequate for inclusion in dairy water buffalo feed. However, the murumuru cake addition requires some caution because its use induces the secretion of higher levels of lauric and myristic fatty acids that are related to human cardiovascular disease.