Conjugated linoleic acid (CLA) modulates bovine peripheral blood mononuclear cells (PBMC) proteome in vitro.

Conjugated linoleic acid (CLA) is a group of natural isomers of the n-6 polyunsaturated fatty acid (PUFA) linoleic acid, exerting biological effects on cow physiology. This study assessed the impact of the mixture 50:50 (vol:vol) of CLA isomers (cis-9, trans-11 and trans-10, cis-12) on bovine peripheral blood mononuclear cells (PBMC) proteome, identifying 1608 quantifiable proteins. A supervised multivariate statistical analysis, sparse variant partial least squares - discriminant analysis (sPLS-DA) for paired data identified 407 discriminant proteins (DP), allowing the clustering between the CLA and controls. The ProteINSIDE workflow found that DP with higher abundance in the CLA group included proteins related to innate immune defenses (PLIN2, CD36, C3, C4, and AGP), with antiapoptotic (SERPINF2 and ITIH4) and antioxidant effects (HMOX1). These results demonstrated that CLA modulates the bovine PBMC proteome, supports the antiapoptotic and immunomodulatory effects observed in previous in vitro studies on bovine PBMC, and suggests a cytoprotective role against oxidative stress. SIGNIFICANCE: In this study, we report for the first time that the mixture 50:50 (vol:vol) of cis-9, trans-11, and trans-10, cis-12-CLA isomers modulates the bovine PBMC proteome. Our results support the immunomodulatory and antiapoptotic effects observed in bovine PBMC in vitro. In addition, the present study proposes a cytoprotective role of CLA mixture against oxidative stress. We suggest a molecular signature of CLA treatment based on combining a multivariate sparse discriminant analysis and a clustering method. This demonstrates the great value of sPLS-DA as an alternative option to identify discriminant proteins with relevant biological significance.

Heat stress has divergent effects on the milk microbiota of Holstein and Brown Swiss cows.

Heat stress (HS) is one of the pivotal causes of economic losses in dairy industries and affects welfare and performance, but its effect on milk microbiota remains elusive. It is also unclear if and how different breeds may cope with HS in sustaining productive performance. The objectives of this study were to compare a) the performance of 2 dairy breeds, namely Holstein and Brown Swiss, subjected to HS and b) the different effects of HS on the milk microbiota of the 2 breeds in thermal comfort conditions and HS. The study was carried out on 36 dairy cows, 18 per breed. The HS was induced by switching off the cooling system during a natural heat wave for 4 d. Besides the Temperature Humidity Index (THI), the animal stress was confirmed by measuring respiratory frequency and rectal temperature twice daily at 4 a.m. and 3 p.m. The HS differently impacted the 2 breeds. Rectal temperatures were higher in Holstein cows, while no changes in rectal temperature were found in Brown Swiss. Milk yield recording and sampling were performed during the morning milking of d 1 (at 4.00 a.m.) and afternoon milking of d 4 (at 5.00 p.m.). Productive parameters were also different: milk yield, fat-corrected milk, energy-corrected milk, protein and casein content, and renneting parameters were decreased in Holstein but remained unaffected in Brown Swiss. The HS also modified the milk microbiota of the 2 breeds differently. During HS, the Brown Swiss milk microbiota was richer (α diversity) than the Holstein one. Comparing the time points before and during HS within breeds showed that Brown Swiss milk microbiota was less affected by HS than Holstein's. Under the same thermal comfort condition, milk microbiota did not discriminate between Brown Swiss and Holstein. Consistently with α and ß diversity, the number of operational taxonomic units (OTUs) at the genus level that changed their abundance during HS was higher in Holstein (74 OTUs) than in Brown Swiss (only 20 OTUs). The most significant changes in abundance affected Acinetobacter, Chryseobacterium, Cutibacterium, Enterococcus, Lactococcus, Prevotella-9, Serratia, and Streptococcus. In conclusion, the present report confirms and extends previous studies by demonstrating that Brown Swiss cows regulate their body temperature better than the Holstein breed. The relative thermal tolerance to HS compared with Holstein is also confirmed by changes in milk uncultured microbiota, which were more evident in Holstein than in Brown Swiss.

The untargeted lipidomic profile of quarter milk from dairy cows with subclinical intramammary infection by non-aureus staphylococci.

This observational study determined the lipidome of cow milk during subclinical intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted approach. Among the pathogens causing bovine IMI, NAS have become the most frequently isolated bacteria from milk samples. Although the application of system biology approaches to mastitis has provided pivotal information by investigating the transcriptome, proteome, peptidome, and metabolome, the milk lipidome during mammary gland inflammation remains undisclosed. To cover this gap, we determined the milk lipidome of 17 dairy cows with IMI caused by NAS (NAS-IMI), and we compared the results with those of healthy quarter milk from 11 cows. The lipidome was determined following a liquid chromatography-quadrupole time-of-flight mass spectrometry approach. Sixteen subclasses of lipids were identified in both groups of animals. From 2,556 measured lipids, the abundance of 597 changed more than 10-fold in quarter milk with NAS-IMI compared with healthy quarters. The results demonstrate the influence of NAS-IMI on the milk lipidome, implying significant changes in lipid species belonging to the family of triacylglycerols and sphingomyelins, and contribute to the understanding of inflammatory processes in the bovine udder, highlighting potential novel biomarkers for improving mastitis diagnostics.

Anti-inflammatory activity of citrus pectin on chicken monocytes' immune response.

Pectin is a dietary fibre composed of galacturonic acid, primarily found in the citrus fruits' cell walls. Citrus pectin (CP) has demonstrated antioxidative, anticancer, and anti-inflammatory properties in humans and animals. In broilers, CP supplementation improves energy utilization and nutrient digestibility, but limited information on its effects on chicken immunity is available so far. This study aimed to assess the in vitro impact of CP on chicken monocytes' immune response. Cells were purified from whole blood of healthy chickens and incubated with increasing concentrations (0, 0.25, 0.5, 0.75, 1 mg/mL) of CP to determine CP working concentration. The effects of different CP concentrations on cells' apoptosis and viability were assessed by measuring caspase-3 and -7 and the cells' metabolic activity (MTT assay), respectively. CP had no dose-dependent effect on monocyte apoptosis and viability.Then, the effects of CP (0.5 mg/mL) on chicken monocytes' chemotaxis and phagocytosis were assessed by measuring transwell migration and fluorescein-labelled E. coli incorporation, respectively. CP inhibited both monocytes' chemotaxis and phagocytosis.These data demonstrate that CP exerts an immunomodulatory role in chicken monocytes, supporting its integration in nutrition strategies that might be beneficial for the animal's immunity and health.

Methods in isolation and characterization of bovine monocytes and macrophages.

Monocytes and macrophages belong to the mononuclear phagocyte system and play important roles in both physiological and pathological processes. The cells belonging to the monocyte/macrophage system are structurally and functionally heterogeneous. Several subsets of monocytes have been previously identified in mammalian blood, generating different subpopulations of macrophages in tissues. Although their distribution and phenotype are similar to their human counterpart, bovine monocytes and macrophages feature differences in both functions and purification procedures. The specific roles that monocytes and macrophages fulfil in several important diseases of bovine species, including among the others tuberculosis and paratuberculosis, brucellosis or the disease related to peripartum, remain still partially elusive. The purpose of this review is to discuss the current knowledge of bovine monocytes and macrophages. We will describe methods for their purification and characterization of their major functions, including chemotaxis, phagocytosis and killing, oxidative burst, apoptosis and necrosis. An overview of the flow cytometry and morphological procedures, including cytology, histology and immunohistochemistry, that are currently utilized to describe monocyte and macrophage main populations and functions is presented as well.

In vitro effects of conjugated linoleic acid (CLA) on inflammatory functions of bovine monocytes.

The conjugated linoleic acid (CLA) isomers, a group of naturally occurring isomers of the essential fatty acid (FA) linoleic acid, have received special attention in animal and human nutrition. Although they have long been used as dietary integrators in dairy cows, the effects of CLA isomers on bovine immune cells remain mostly undisclosed. The present study aimed to cover this gap and investigate the in vitro effects of CLA on inflammatory functions, including chemotaxis, phagocytosis, killing capability, and extracellular respiratory burst of purified bovine monocytes (CD14+). The apoptosis rate of monocytes was addressed as well. Once assessed, the effects of different concentrations (10, 50, 100, and 500 µM) of the 2 main CLA isomers, namely cis-9,trans-11 and trans-10,cis-12, the experiments were carried out using a concentration of 50 µM of the CLA isomers, both individually and in a mixture (50:50). The immunomodulatory activities of linoleic acid, an essential FA, and stearic acid, a saturated FA, were also investigated. Only the 50:50 CLA mixture was able to reduce monocyte apoptosis and to increase the extracellular respiratory burst during experimental proinflammatory conditions, as assessed by measuring production of reactive oxygen species. Linoleic acid and CLA had no effects on chemotaxis, phagocytosis, or killing capability. Remarkably, treatment of monocytes with stearic acid significantly reduced their chemotactic capability. The present results demonstrated that CLA isomers do have immunomodulatory effects on some functions of bovine monocytes, and that the mixture of the 2 CLA isomers is more effective than the CLA isomers individually.

Circulating miR-23b-3p, miR-145-5p and miR-200b-3p are potential biomarkers to monitor acute pain associated with laminitis in horses.

Circulating microRNAs (miRNAs) are emerging as promising biomarkers for several disorders and related pain. In equine practice, acute laminitis is a common disease characterised by intense pain that severely compromises horse welfare. Recently, the Horse Grimace Scale (HGS), a facial expression-based pain coding system, was shown to be a valid welfare indicator to identify pain linked to acute laminitis. The present study aimed to: determine whether miRNAs can be used as biomarkers for acute pain in horses (Equus caballus) affected by laminitis; integrate miRNAs to their target genes and to categorise target genes for biological processes; gather additional evidence on concurrent validity of HGS by investigating how it correlates to miRNAs. Nine horses presenting acute laminitis with no prior treatment were recruited. As control group, nine healthy horses were further included in the experimental design. Samples were collected from horses with laminitis at admission before any treatment ('pre-treatment') and 7 days after routine laminitis treatment ('post-treatment'). The expression levels of nine circulating miRNAs, namely hsa-miR-532-3p, hsa-miR-219-5p, mmu-miR-134-5p, mmu-miR-124a-3p, hsa-miR-200b-3p, hsa-miR-146a-5p, hsa-miR-23b-3p, hsa-miR-145-5p and hsa-miR-181a-5p, were detected and assessed as potential biomarkers of pain by quantitative PCR using TaqMan® probes. The area under the receiver operating curve (AUC) was then used to evaluate the diagnostic performance of miRNAs. Molecular data were integrated with HGS scores assessed by one trained treatment and time point blind veterinarian. The comparative analysis demonstrated that the levels of miR-23b-3p (P=0.029), miR-145-5p (P=0.015) and miR-200b-3p (P=0.023) were significantly higher in pre-treatment and the AUCs were 0.854, 0.859 and 0.841, respectively. MiR-200b-3p decreased after routine laminitis treatment (P=0.043). Combining two miRNAs in a panel, namely miR-145-5p and miR-200b-3p, increased efficiency in distinguishing animals with acute pain from controls. In addition, deregulated miRNAs were positively correlated to HGS scores. Computational target prediction and functional enrichment identified common biological pathways between different miRNAs. In particular, the glutamatergic pathway was affected by all three miRNAs, suggesting a crucial role in the pathogenesis of pain. In conclusion, the dynamic expression of circulating miR-23b-3p, miR-145-5p and miR-200b-3p was detected in horses with acute laminitis and miRNAs can be considered potentially promising pain biomarkers. Further studies are needed in order to assess their relevancy in other painful conditions severely compromising horse welfare. An important implication would be the possibility to use them for the concurrent validation of non-invasive indicators of pain in horses.

Application of post-genomic techniques in dog cancer research.

Omics techniques have been widely applied to veterinary science, although mostly on farm animal productions and infectious diseases. In canine oncology, on the contrary, the use of omics methodologies is still far behind. This review presents the most recent achievement in the application of postgenomic techniques, such as transcriptomics, proteomics, and metabolomics, to canine cancer research. The protocols to recover material suitable for omics analyses from formalin-fixed, paraffin-embedded tissues are presented, and omics applications for biomarker discovery and their potential for cancer diagnostics in veterinary medicine are highlighted.

Circulating extracellular miR-22, miR-155, and miR-365 as candidate biomarkers to assess transport-related stress in turkeys.

MicroRNA (miRNA) have been identified in circulating blood and might have the potential to be used as biomarkers for several pathophysiological conditions. To identify miRNA that are altered following stress events, turkeys (Meleagris gallopavo) were subjected to 2 h of road transportation. The expression levels of five circulating miRNA, namely miR-22, miR-155-5p, miR-181a-3p, miR-204 and miR-365-3p, were detected and assessed by quantitative polymerase chain reaction using TaqMan® probes, as potential biomarkers of stress. The areas under the receiver operating characteristic curves were then used to evaluate the diagnostic performance of miRNA. A panel of three stress-responsive miRNA, miR-22, miR-155 and miR-365 were identified; their expression levels were significantly higher after road transportation and the area under the curve (AUC) were 0.763, 0.71 and 0.704, respectively. Combining the three miRNA a specificity similar to the one found for the three miRNA separately was found. The AUC of the weighted average of the three miRNA was 0.763. This preliminary study suggests that the expression levels of circulating miR-22, miR-155 and miR-365 are increased during transport-related stress and that they may have diagnostic value to discriminate between stressed- and unstressed animals.

UCP1 and UCP2 expression in different subcutaneous and visceral adipose tissue deposits in 30 days old goat kids and effect of fatty acid enriched diets.

Beside its importance in the first hours of life, brown adipose tissue has also significant roles in the following stages of growth and in adults by regulating energy metabolism, but its identification in adult ruminants is still controversial. Quantitative PCR, followed by histological confirmation, was used to investigate UCP expression and brown and white adipocytes' distribution in 30-day-old goat kids. The influence of maternal diet enriched with either fish oil or stearic acid was investigated as well. Results showed the differential expression of both UCP1 and UCP2 genes between subcutaneous and visceral adipose tissues, suggesting a different thermogenic activity between the two macro areas. The maternal diet influenced neither UCP1 nor UCP2 gene expression. The presence of multilocular adipocytes in 1-month goat kids is remarkable, as suggests thermogenic activity in non-newborn animals. Further insights into characteristics and functions of adipose tissue in young and adult goats are worth exploring.

Animal board invited review: advances in proteomics for animal and food sciences.

Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid - i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002--Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East-West and North-South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.

Proteomics in veterinary medicine: applications and trends in disease pathogenesis and diagnostics.

Advancement in electrophoresis and mass spectrometry techniques along with the recent progresses in genomics, culminating in bovine and pig genome sequencing, widened the potential application of proteomics in the field of veterinary medicine. The aim of the present review is to provide an in-depth perspective about the application of proteomics to animal disease pathogenesis, as well as its utilization in veterinary diagnostics. After an overview on the various proteomic techniques that are currently applied to veterinary sciences, the article focuses on proteomic approaches to animal disease pathogenesis. Included as well are recent achievements in immunoproteomics (ie, the identifications through proteomic techniques of antigen involved in immune response) and histoproteomics (ie, the application of proteomics in tissue processed for immunohistochemistry). Finally, the article focuses on clinical proteomics (ie, the application of proteomics to the identification of new biomarkers of animal diseases).

Hepatic and extrahepatic expression of serum amyloid A3 during lactation in dairy cows.

Serum amyloid A3 (SAA3) is the predominant SAA isoform secreted by mammary epithelial cells in dairy cows; it is also expressed in bovine adipose tissue (AT). The adipokine SAA3 is linked to obesity and insulin resistance of AT and the respective inflammatory response, at least in mice. Dietary treatment with conjugated linoleic acids (CLA) reportedly also affects insulin sensitivity and inflammatory status in monogastrics. Both SAA3 and CLA thus seem to alter similar functions. Based on changes in insulin sensitivity and the inflammatory status throughout lactation, we hypothesized that the mRNA abundance of SAA3 in various tissues might be regulated as well and that CLA could be a modulator of SAA3 mRNA expression. In 2 trials, 21 pluriparous and 25 primiparous Holstein cows were fed 100g/d of a CLA or a control fat supplement from d 1 to 182 or 105 postpartum, respectively. Biopsies from liver and subcutaneous (s.c.) AT from pluriparous cows and samples from 3 different visceral AT and 3 s.c. AT, muscle, mammary gland, and liver tissue from slaughtered primiparous cows were obtained. In an adipocyte cell culture system, cell samples were collected during differentiation of bovine preadipocytes at d 0, 2, 6, 8, 10, 12, and 13 relative to the onset of differentiation. The SAA3 mRNA abundance in tissues and in differentiating bovine preadipocytes was measured by real-time PCR. The presence of the SAA protein was confirmed by Western blotting. Treatment with CLA yielded only few and inconsistent effects on SAA3 mRNA abundance. In both trials, SAA3 mRNA peaked at d 1 postpartum in all tissues except in mesenteric AT, in which the change was not significant. The highest SAA3 mRNA expression was observed in the mammary gland, followed by omental AT. The SAA protein was present in the visceral and s.c. AT depots investigated. Adipocytes as one source of SAA3 were confirmed by the SAA3 mRNA profile in differentiating adipocytes. The longitudinal changes observed point to SAA3 being involved in the inflammatory situation around parturition.

Hyposialylated α1-acid glycoprotein inhibits phagocytosis of feline neutrophils.

Feline α1-acid glycoprotein (fAGP) modifies both its serum concentration and its glycan moiety during diseases. fAGP is hyposialylated in cats with feline infectious peritonitis (FIP), but not in clinically healthy cats or in cats with other diseases. This study was aimed to determine whether hyposialylated fAGP influences phagocytosis. A flow cytometric method based on ingestion of fluoresceinated bacteria and adapted to feline blood was used to assess phagocytosis of leukocytes incubated with 'non-pathological' fAGP (purified from sera with normal concentrations of AGP) and 'pathological' fAGP (purified from sera with >1.5mg/mL hyposialylated AGP). The flow cytometric method provided repeatable results for neutrophils (coefficients of variations, CVs <15%) but not for monocytes (CVs>20%) which had also a high individual variability. Compared with saline solution and with non-pathological fAGP, pathological fAGP significantly decreased phagocytosis in neutrophils and monocytes. This study demonstrated that hyposialylated fAGP down-regulates the phagocytic activity of feline neutrophils.

Acute phase proteins in ruminants.

The physiological response to infections and injuries involves local inflammation and the initiation of events leading to a systemic response, also called acute phase reaction (APR). This multiplicity of changes is distant from the site of injury, and includes fever, leukocytosis and quantitative and qualitative modification of a group of non-structurally related proteins present in blood and other biological fluids, collectively named Acute Phase Proteins (APP). Proteomic investigations of serum or plasma following natural or experimental infection frequently reveal substantial alterations in the APP, several of which are high abundance proteins in these fluids. The present review will focus on the results of recent research on ruminant APP. Highlight points will include: - The structure and the functions of the main APPs in ruminants, as well as the regulatory mechanisms that trigger their systemic and local expression in both physiological and pathological conditions.- The clinical aspects of APPs in ruminants, including the current and future application to veterinary diagnosis and animal production.- The APP in small and wildlife ruminants.- Alteration in APP detected by proteomic investigations.

TIR8 receptor expression in bovine tissues.

The TIR8 receptor (also called SIGIRR) is an orphan member of the TIR superfamily. Its function is still elusive, but it is believed to trigger a negative pathway of regulation of the Toll-like/IL-1 receptor system, crucial for modulating inflammation in gastrointestinal (GI) tract and in other tissues (lung and kidney). The expression pattern of TIR8 in bovine tissues is unknown. Given the importance of GI diseases in cattle, the aim of this investigation was to study the distribution of TIR8 in a wide panel of non-pathologic tissues and organs. TIR8 expression was assessed by Northern blot analysis and further confirmed and comparatively quantified by qualitative and quantitative (Real-Time) PCR. The possible presence of tissue-specific isoforms was determined by Western blot immunodetection, using an anti-human TIR8 polyclonal antibody previously validated in bovine tissues. Similarly to humans and mice, bovine TIR8 was found in the GI tract and kidney. Expression of TIR8 mRNA was also detected in lymph nodes, thymus and thyroid gland. Interestingly, several isoforms of bovine TIR8 were detected in the same organs, suggesting the occurrence of different post-translational processings.

FlgM anti-sigma factors: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence-structure-function relationships.

FlgM proteins, also known as Anti-sigma-28 factor (sigma28), are negative regulators of flagellin synthesis. Recently, a three-dimensional structure of the Aquifex aeolicus sigma28/FlgM complex (PDB code: 1rp3) was determined by X-ray crystallography at 2.3 A resolution. Furthermore, experimental data on bacterial FlgM, including site-directed mutagenesis and structural characterization by NMR are also available. However, an interpretation of the sequence-structure-function relationships combining X-ray and NMR data with the evolutionary information extracted from the increasing number of FlgM-related sequences annotated in databases is not available. In the present study, we combined database sequence searches and sequence-analysis tools to update the multiple sequence alignment of a previously characterized cluster of orthologs (COG2747) and the PFAM classification of protein domains (PF04316) for the FlgM family. A phylogenetic analysis of 77 protein sequences revealed the presence of at least three major sequence clades within the FlgM family. Besides, we predicted functional residues using a SequenceSpace method. We also generated homology models for Bacillus subtilis and Salmonella typhimurium FlgM proteins, for which sequence-structure-function relationship data are available, and used the docking program ClusPro to hypothesize about the dimer association between FlgM proteins. In conclusion, the analysis presented in this work will be useful in designing new experiments to understand better protein-protein interactions between FglM, sigma factors, and putative molecules from the flagellar export apparatus. Electronic Supplementary Material is available in the online version of this article at http://link.springer.de/

Tissue distribution of a feline AGP related protein (fAGPrP) in cats with feline infectious peritonitis (FIP).

Feline alpha(1)-acid glycoprotein (fAGP) increases during feline infectious peritonitis (FIP). We have recently identified a 29 kDa protein that we named feline AGP-related protein (fAGPrP) due to its cross-reactivity with an anti-human AGP monoclonal antibody. In this work we describe the tissue distribution of fAGPrP during FIP, and its relationship with feline coronavirus (FCoV) and myeloid cells. Tissues from five control cats and from 15 cats with FIP were examined by immunohistochemistry using monoclonal antibodies against human AGP, FCoV and myeloid antigens. Diffuse fAGPrP positivity within the lesions, likely due to vascular plasma leakage, endothelial and epithelial lining were detectable. Compared to controls, fAGPrP-expressing cells often increased in number and were diffusely distributed in lymph nodes, as usually occurs for IgM-producing plasma cells during early immune responses. These findings did not depend on the presence of FCoVs or of myeloid cells, suggesting that fAGPrP is not directly involved in the pathogenesis of FIP.

Feline dirofilariosis: antibody response to antigenic fractions containing specific 20 to 30 kDa polypeptides from the adult Dirofilaria immitis somatic antigen.

Fractions from the adult somatic antigen (SA) Dirofilaria immitis complex, containing polypeptides from 20 to 30kDa, previously identified as molecular markers of feline dirofilariosis are isolated by sequential application of gel filtration and anion exchange chromatography. Indirect enzyme-linked immunosorbent assays, employing these fractions (20-26kDa/ELISAF1 and 30kDa/ELISAF7) show multivalent diagnostic capacities: they were able to detect pre-patent infections 2 months after infection, infections in clinical phase, and the fall of antibodies after the worms were removed from the heart, or the application of a ivermectin treatment. The results obtained by the two tests correlated well, in spite of the fact that ELISAF1 was most useful to detect antibodies in sera from cats in the clinical phase, while ELISAF7 has more sensitivity for the early detection of the infections. Both ELISAs were useful in the detection of the decrease of antibodies after the worms were removed by surgery or pharmacological treatment.

Cloning, sequencing and expression in the seeds and radicles of two Lupinus albus conglutin gamma genes.

Two genes encoding conglutin gamma have been isolated from a Lupinus albus genomic library and sequenced. The expression of conglutin gamma was studied by partial amino acid sequencing of the mature seed protein and by nucleotide sequencing of reverse transcriptase-polymerase chain reaction products from various tissues during the plant life cycle.