Evaluation of two cocktails containing ESAT-6, CFP-10 and Rv-3615c in the intradermal test and the interferon-γ assay for diagnosis of bovine tuberculosis

The intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay are the principal tests used worldwide for the ante-mortem diagnosis of bovine tuberculosis. The conventional reagent currently in use in these tests is purified protein derivative (PPD) tuberculin obtained from Mycobacterium bovis culture. The components of PPD are poorly characterized and difficult to standardize. To overcome this issue, antigens specific to the Mycobacterium tuberculosis complex are being studied. Here we have assessed the biological potency of ESAT-6, CFP-10 and Rv-3615c presented as peptide or recombinant protein cocktails in comparison with the standard bovine PPD used routinely in Spanish eradication campaigns. The study was performed in cattle (n=23) from a herd with natural M. bovis infection. Animals were simultaneously injected with PPD and the peptide and protein cocktails. The percentages of cattle reacting positively to single intradermal test were 60.9% (bovine PPD), 47.8% (peptide cocktail) and 60.9% (protein cocktail), with no significant difference between the actual skin fold thickness increases (p>0.05). The IFN-γ assay detected 60.9% of animals when stimulation was performed with bovine PPD, but decreased to 52.2% when stimulation was performed with the peptide cocktail and to 47.8% when stimulation was performed with the protein cocktail. However, no significant differences were found between IFN-γ responder frequencies (p>0.05). These results show a potential use of these defined reagents for in vivo tuberculosis diagnosis.     

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.     

Evaluation of a fluorescence-polarization assay for the diagnosis of bovine brucellosis in México.

A homogeneous fluorescence-polarization assay (FPA) was used for the serological diagnosis of bovine brucellosis in México. The assay uses O-polysaccharide prepared from Brucella abortus lipoplysaccharide (20-30 kDa) conjugated with fluorescein isothiocyanate as a tracer. To measure the fluorescence polarization, a FPM-1 fluorescence-polarization analyzer was used with the procedure described by Nielsen et al. (1996b). A cut-off value of 90 millipolarization (mP) units was used for testing 560 bovine sera from different areas of México. (305 positive sera and 255 negative sera according to the complement fixation test; CFT.) Some were tested with the Rose Bengal plate (RB) test (n = 490) and some with the rivanol-agglutination (RIV) test (n = 190). Sensitivities were 98.3%, 99.3% and 99.0%, and specificities were 68.8%, 55.4% and 96.9%, respectively, for RB, RIV and FPA. The FPA gave a kappa coefficient of agreement with respect to CFT of 0.96, while RB and RIV (relative to the CFT) gave coefficients of 0.70 and 0.61, respectively. Finally, ROC analysis suggested a cut-off value which agreed with the one recommended in the test procedure. We concluded that FPA is a suitable test to be used instead of the CFT in Mexican conditions.     

Comparative study of IFNγ and antibody tests for feline tuberculosis

This study describes the comparison of the cell-based interferon-gamma (IFNγ) test with serological rapid antibody tests (STAT-PAK and DPP VetTB) for the ante mortem testing of tuberculosis in domestic cats. The antibody specificities of rapid antibody test-positive cats were further discerned using multi-antigen print immunoassay. A total of 62 cats with culture-confirmed Mycobacterium bovis, Mycobacterium microti, Mycobacterium avium and Mycobacterium malmoense, as well as negative controls and dangerous-contact cats were tested. Tests were also applied longitudinally to one further cat undergoing TB chemotherapy for suspected M. bovis infection. Our data from this small study show excellent test specificity (100% for all tests) and encouraging levels of test sensitivity for M. bovis and TB Complex infections (IFNγ 70-100% depending upon test interpretation criteria; rapid tests both 90% for M. bovis infection and up to 46.2% for M. microti infection). The differential diagnosis of very pathogenic TB Complex (M. bovis, Mycobacterium tuberculosis), as opposed to less-pathogenic TB Complex (M. microti) was possible where positive responses to the protein cocktail ESAT6/CFP10 were observed (80% of M. bovis-infected cats in this study showed positive IFNγ responses to ESAT6/CFP10, while 20% had antibody responses to ESAT6/CFP10 using MAPIA). Finally, preliminary data from a longitudinal study of one M. bovis-exposed cat with a positive IFNγ test pre-treatment suggest that a decrease in bacterial burden may be reflected in the IFNγ response, and thus the IFNγ test may provide a monitor for TB chemotherapy.     

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.     

Immunohistochemical expression of interferon-γ in different types of granulomatous lesions associated with bovine paratuberculosis

Animals infected with Mycobacterium avium subsp. paratuberculosis (Map) show a variety of lesions, from focal forms, seen in subclinical stages to diffuse lesions in clinical cases. The purpose of this study was to evaluate the local expression of IFN-γ by immunohistochemistry in relation with the type of lesion in naturally Map-infected cows. The number of immunolabelled cells, −the majority morphologically consistent with lymphocytes-, was higher in focal and diffuse paucibacillary forms than in diffuse multibacillary lesions, where they appeared closely related to epithelioid cells. Diffuse multibacillary lesions had the lowest numbers, but higher than controls, and positive cells were intermingled among the macrophages. The peripheral IFN-γ production was higher in all Map infected cows and a positive correlation was found with the number of immunolabelled cells in the intestine. The findings of this study show that IFN-γ would play a role in the development of the different types of lesions in paratuberculosis, and also points out the importance of adequate sampling of lymphoid tissue containing samples when studying the local immune response in which IFN-γ expression may be involved, especially in cases where focal lesions are present.     

Effects of interferon-γ knockdown on vaccine-induced immunity against Marek’s disease in chickens

Interferon (IFN)-γ has been shown to be associated with immunity to Marek’s disease virus (MDV). The overall objective of this study was to investigate the causal relationship between IFN-γ and vaccine-conferred immunity against MDV in chickens. To this end, 3 small interfering RNAs (siRNAs) targeting chicken IFN-γ, which had previously been shown to reduce IFN-γ expression in vitro, and a control siRNA were selected to generate recombinant avian adeno-associated virus (rAAAV) expressing short-hairpin small interfering RNAs (shRNAs). An MDV challenge trial was then conducted: chickens were vaccinated with herpesvirus of turkey (HVT), administered the rAAAV expressing shRNA, and then challenged with MDV. Tumors were observed in 4 out of 10 birds that were vaccinated with HVT and challenged but did not receive any rAAAV, 5 out of 9 birds that were administered the rAAAV containing IFN-γ shRNA, and 2 out of 10 birds that were administered a control enhanced green fluorescent protein siRNA. There was no significant difference in MDV genome load in the feather follicle epithelium of the birds that were cotreated with the vaccine and the rAAAV compared with the vaccinated MDV-infected birds. These results suggest that AAAV-based vectors can be used for the delivery of shRNA into chicken cells. However, administration of the rAAAV expressing shRNA targeting chicken IFN-γ did not seem to fully abrogate vaccine-induced protection.     

Biology of fibre growth and possible genetic and non-genetic means of influencing fibre growth in sheep and goats—a review

This paper reviews the biology of wool, mohair and cashmere growth and the options available to sheep and goat farmers to manipulate the quantity and characteristics of fibre produced to meet processors' requirements. The annual pattern of fibre growth in sheep and fibre producing goats is photodependent and regulated by hormone action. In sheep and Angora goats, fibre growth rate is greater in summer than winter with the amplitude varying between breeds. Cashmere-producing goats grow fibre between the longest and shortest day after which it is shed. The large diversity in fleece and fibre characteristics between genotypes of sheep and goats can be used to change characteristics by selection. Fibre characteristics of greatest importance to processors are all of medium to high heritability. Negative genetic correlations between some important characteristics limit overall genetic progress achievable through selection. The principal non-genetic factors affecting fleece and fibre characteristics in both sheep and goats are age, nutrition, physiological status, disease and shearing regime. Further work is required to gain a greater understanding of biological changes occurring within the follicle and fibre during growth to facilitate the production of fibre of specific characteristics through breeding or altering animal management.     

Development of an Heal amino acid digestibility assay for the growing chicken—effects of time after feeding and site of sampling

1. The study aimed to establish the optimum time after ingestion and optimum sampling site for the development of an ileal amino acid digestibility assay for broiler chickens.

2. To establish the optimal sampling time, 4‐week‐old broiler chickens were given one of 6 protein sources (meat‐and‐bone, soyabean, cottonseed, fish, maize and wheat meals) as the sole source of protein in a test diet. The diets contained chromic oxide as an indigestible marker. The birds were starved for 24 h, fed and subsequently killed for sampling of ileal digesta (terminal 15 cm) at 2, 3, 4, 5 and 6 h after the start of feeding.

3. For the soyabean, fish, wheat and maize meal diets, sampling time had no significant effect on apparent ileal nitrogen digestibility, whereas for the meat‐and‐bone and cottonseed meal diets there was a significant quadratic effect of sampling time. The amount of digesta collected was maximised and the mean apparent ileal nitrogen digestibility had the lowest variation around the 4 h sampling time.

4. To establish the optimum sampling site, 4‐week‐old chickens were given either a meat‐and‐bone, a soyabean or a wheat bran meal‐based diet. The birds were killed 4 h after the start of feeding, and digesta were sampled from 0.10, 0.15, 0.20 or 0.25 cm of terminal ileum.

5. There was no significant effect of sampling site on the apparent ileal digestibility of dietary nitrogen. The terminal 15 cm of ileum was considered a preferred site for sampling ileal digesta from broiler chickens.     

Induction of antibody and interferon-γ production in mice immunized with virus-like particles of swine hepatitis E virus

Virus-like particles (VLPs) composed of the truncated capsid protein of swine hepatitis E virus (HEV) were developed and immune responses of mice immunized with the VLPs were evaluated. IgG titers specific for the capsid protein of swine HEV were significantly higher for all groups of mice immunized with the VLPs than those of the negative control mice. Splenocytes from mice immunized with the VLPs also produced significantly greater quantities of interferon (IFN)-γ than interleukin (IL)-4 and IL-10. These newly developed swine HEV VLPs have the capacity to induce antigen-specific antibody and IFN-γ production in immunized mice.     

Test protocol of an enzyme‐linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukosis virus

Summary

In this communication the test procedure is described for an indirect enzyme‐linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukosis virus (BLV). Test sera are incubated in polystyrene microtiter plates sensitized with a partly‐purified preparation of BLV. Bovine antibodies are detected with anti‐species immunoglobulin conjugated to the enzyme horseradish peroxidase, followed by the addition of the enzyme substrate.     

Interferon-γ acts as a regulator in the trade-off between phagocytosis and production performance in dwarf chickens

Background: Interferon-γ(IFN-γ) is critical for innate and adaptive immunity against viral and bacterial infections. IFN-γ reportedly affects the phagocytic ability of monocytes and macrophages as well as regulates pituitary function in humans and mice. The present study analyzed the impact of IFN-γ on monocyte and macrophage phagocytosis, production performance, and pituitary function in vivo and in vitro(in dwarf chickens). IFN-γ was injected into dwarf chickens through a vein, and then, the laying rate, average egg weight, and levels of follicle-stimulating hormone(FSH) and IFN-γ were measured in treatment and control groups. For the in vitro experiment, the pituitary tissues were supplemented with IFN-γ, and the m RNA expression levels of follicle-stimulating hormone beta subunit(FSH-β), interferon gamma receptor 1(IFNGR1),and interferon gamma receptor 2(IFNGR2) in the pituitary were assessed.Results: Monocyte and macrophage phagocytosis product(PP) was decreased by IFN-γ treatment in a dose-dependent manner in vitro. In the in vivo experiment, the level of IFN-γ in the treatment group was higher than that in the control group at 7 d(P 0.05), 14 d(P 0.01), and 21 d(P 0.01) post-injection.Compared with the control group, monocyte and macrophage PP was lower in the treatment group after injection(P 0.01). The laying rate was higher in the treatment group than in the control group at 2 and3 wk post-injection(P 0.05). There was a significant difference between the treatment and control groups in the levels of FSH at 1, 3, 7, and 14 d post-injection(P 0.01). In the in vitro experiment, increased m RNA expression levels of FSH-β, IFNGR1, and IFNGR2 were observed in the treatment group after stimulation with100 U/m L IFN-γ for 24 h compared to those in the control group(P 0.05).Conclusions: IFN-γ inhibited the phagocytosis of monocytes and macrophages; up-regulated the m RNA expression levels of the FSH-β, IFNGR1, and IFNGR2; enhanced the secretion of FSH; and improved the laying rate. IFN-γ might be an important regulator in the trade-off between the immune effect and production performance in dwarf chickens.     

Point-of-care β-hydroxybutyrate measurement for the diagnosis of feline diabetic ketoacidaemia

Objectives : To evaluate accuracy and precision of a hand‐held ketone meter measuring β‐hydroxybutyrate and to determine its diagnostic performance to rule out ketoacidaemia in diabetic cats. Methods : The ketone meter was validated by calculating within‐day precision at different β‐hydroxybutyrate concentrations and by comparison with a laboratory method. To determine its diagnostic performance to diagnose ketoacidaemia, 217 sets of data (venous blood gas analysis and β‐hydroxybutyrate measurements) were retrospectively analysed. Sensitivities and specificities were calculated with the help of receiver‐operating characteristic curves. Results : The ketone meter reliably detected β‐hydroxybutyrate at concentrations >0·1 mmol/L and reproducibility was acceptable. Measurements highly correlated with laboratory results (r=0·97; P<0·001), but a significant negative bias was found at high concentrations. A β‐hydroxybutyrate concentration of >2·55 mmol/L had a sensitivity of 94% and a specificity of 68% for diagnosing ketoacidaemia. Many cats with high β‐hydroxybutyrate concentrations and normal blood pH had an elevated chloride gap suggestive of superimposed hypochloraemic metabolic alkalosis. Clinical Significance : The commercially available point‐of‐care ketone meter Precision Xtra is a valid tool to measure β‐hydroxybutyrate in diabetic cats. Concentration <2·55 mmol/L enable ketoacidaemia to be excluded and should lead to redirection of differential diagnoses.     

Ovarian response and embryo yield of Angora and Kilis goats given the day 0 protocol for superovulation in the non-breeding season

The aim of this study was to compare ovarian response and embryo yield of Day 0 protocol in Angora goats (AG) and indigenous Kilis goats (KG) in the non-breeding season. A total of 16 Angora goats (AG group) and 11 Kilis goats (KG group) were used in this study. In the synchronization process, after controlled internal drug release withdrawal, when estrus signs were observed, natural mating was performed. Ovarian response was determined by synchronized laparotomy 6 days after natural mating, and number of corpora lutea (CL) was recorded. Embryos were collected and morphologically evaluated by stereomicroscope. Synchronization rates did not differ between AG (88%, 14/16) and KG group (91%, 10/11). In AG and KG groups, the proportion of CL on the right (44% and 53%, respectively) and left (56% and 47%, respectively) ovaries were similar. The CL number per animal did not differ significantly between the two breeds and was determined as 4.4 ± 0.90 in AG group and 6.4 ± 1.44 in KG group. Transferable embryo yields were significantly higher in AG group (31/42, 74%) compared to KG group (16/46, 35%) in the non-breeding season (P < 0.01). In conclusion, it is suggested that the day 0 protocol can be used for goat superovulation in the non-breeding season; however, transferable embryo yields are affected by the breed.     

The value of Leucaena leucocephala bark in leucaena—grass hay diets for Thai goats

The study assessed the value of Leucaena leucocephala bark in leucaena—grass hay diets fed to Thai goats. Thai goats in metabolism pens were fed diets containing leucaena leaf (55%) + pangola grass hay (hay, 45%); leucaena leaf (48%) + leucaena bark (9%) + hay (43%); leucaena bark (57%) + hay (43%); and hay only. Feed percentages are expressed on a dry weight basis. The digestibilities of dry matter (DM) and crude protein (CP) were measured for the four diets. Leucaena bark had lower CP concentration than the leaf (11.7 vs. 25.9), and the leucaena bark + hay diet had lower DM and CP digestibility than the other diets. The calculated bark digestibilities of DM and CP of 44.1% and 38.2%, respectively, were much lower than the values for the leucaena leaf of 62.9% and 89.1%, respectively. The lower than expected CP digestibility was attributed to higher tannin levels in the bark compared to the leaves. Despite this, the bark was well accepted by the goats and was often preferred to the hay. Stripping of the bark by goats also results in stems that dry quicker and have higher calorific value as fuel. However, if leucaena branches are fed as a sole diet, the goats may consume up to 30% of bark on a DM basis and this would reduce nutritive value and animal productivity.     

Milk yield and composition of crossbred Sahelian × Anglo-Nubian goats in the semi-intensive system in Mali during the preweaning period

The aim of this study was to evaluate milk yield and its composition during the preweaning period for Sahelian goats (SG) and Anglo-Nubian (AN) crossbred depending on some factors. The experiments were conducted from January to December 2008 for 44 suckled and hand-milked does, randomized, and divided into two equal groups: SG (n?=?22) and F₁ Anglo-Nubian × Sahelian goats (1/2AN; n?=?22). The does and their offsprings were kept in a pen where they stayed indoors for 45 days before they were allowed outdoors when the weather was suitable. Each category received supplemental feeds depending on the season (rainy season, dry cold season, and dry hot season). The average daily milk yield was recorded weekly from parturition to 100 days of age. Individual milk samples were taken for chemical analysis in connection with the yield measurements twice per month from the fourth week of lactation throughout the different seasons (rainy, cold dry, and hot dry). The daily milk yield differed between breed types (P?=?0.001) during the preweaning, while the effect of kids' sex on daily milk production was not significant. Litter size affected milk yield up to day 60 (P?=?0.032) where does with twins producing more milk than those with single kid. However, at day 100, both groups had similar (P?=?0.001) milk production. Total milk yield at weaning increased by 103 % in 1/2AN over SG. The highest concentration of total solids of milk was (12.76 %) recorded in the hot dry season. The results of this study indicate that crossbreeding native Sahelian goats with high potential Anglo-Nubian buck improved milk production and its composition.