Isoenzymatic characterization of Phlebotomus ariasi and P. perniciosus of canine leishmaniasis foci from Eastern Pyrenean regions and comparison with other populations from Europe.

An entomological survey was carried out in 2007 in two Pyrenean counties of Lleida province (north-eastern Spain), where cases of autochthonous canine leishmaniasis have been recently reported. Phlebotomus ariasi and P. perniciosus, vectors of Leishmania infantum in the Mediterranean area, were captured. The aim of the present study was to compare these phlebotomine populations with others captured in known leishmaniasis foci in Europe. Populations of these species were studied by analysing the polymorphism of seven enzymatic systems (HK, PGI, PGM, MDH, 6PGD, FUM and ACO) and compared with other specimens from endemic regions of France, Italy, Malta, Portugal and Spain captured in other campaigns, and also with previously published results. Phlebotomus ariasi was more polymorphic than P. perniciosus. Only the ACO locus had diagnostic alleles, but some other alleles show high characteristic frequencies for each species. The neighbour-joining trees separated two population groups in both species. On the basis of the isoenzyme study results, sand fly populations of the Pyrenean region in Lleida province are closely related to those of other nearby leishmaniasis endemic regions in France and Spain.

Immunoproteomic analysis of the protein repertoire of unsporulated Eimeria tenella oocysts.

The apicomplexan protozoans Eimeria spp. cause coccidioses, the most common intestinal diseases in chickens. Coccidiosis is associated with significant animal welfare issues and has a high economic impact on the poultry industry. Lack of a full understanding of immunogenic molecules and their precise functions involved in the Eimeria life cycles may limit development of effective vaccines and drug therapies. In this study, immunoproteomic approaches were used to define the antigenic protein repertoire from the total proteins of unsporulated Eimeria tenella oocysts. Approximately 101 protein spots were recognized in sera from chickens infected experimentally with E. tenella. Forty-six spots of unsporulated oocysts were excised from preparative gels and identified by matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF-MS) and MALDI-TOF/TOF-MS. For unsporulated oocysts, 13 known proteins of E. tenella and 17 homologous proteins to other apicomplexan or protozoan parasites were identified using the 'Mascot' server. The remaining proteins were searched against the E. tenella protein sequence database using the 'Mascot in-house' search engine (version 2.1) in automated mode, and 12 unknown proteins were identified. The amino acid sequences of the unknown proteins were searched using BLAST against non-redundant sequence databases (NCBI), and 9 homologous proteins in unsporulated oocyst were found homologous to proteins of other apicomplexan parasites. These findings may provide useful evidence for understanding parasite biology, pathogenesis, immunogenicity and immune evasion mechanisms of E. tenella.

Changes in the protein secondary structure of hen's egg yolk determined by Fourier transform infrared spectroscopy during the first eight days of incubation.

In this study, incubation-induced alterations in the protein secondary structures of egg yolk and its major fractions (granules, plasma, and low-density lipoproteins [LDL]) were monitored during the first 8 d of embryogenesis using Fourier transform infrared spectroscopy (FTIR) and isoelectric focusing (IEF). Two factors potentially connected with egg yolk protein secondary structure changes were evaluated, i.e., the pH value of incubated egg yolk, and phosvitin, an important egg yolk protein assumed to play an important role in hematopoiesis as the iron carrier during early embryogenesis. However, neither the significant increase in pH value (6.07 to 6.92) of egg yolk during incubation of fertilized eggs, nor the release of iron from phosvitin were found to be directly related to the changes in protein secondary structure in egg yolk and its fractions. FTIR showed that the protein conformation in whole egg yolk, granules, and LDL was stable during incubation, but separate evaluation of the plasma fraction revealed considerable changes in secondary structure. However, it is unlikely that these changes were provoked by structure changes of the proteins originally present in plasma; instead, the physiological influx of albumen into the yolk sac was expected to play an important role in the protein modifications of egg yolk, as was shown both by FTIR and IEF of the water-soluble egg yolk proteins. Moreover, FTIR was used to determine the naturally occurring proportions (%) of the secondary structure elements in egg yolk and its 3 fractions on d 0 of incubation. The granules fraction mainly consisted of a mixture of inter- and intramolecular ß-sheets (57.04%±0.39%). The plasma fraction was found to consist mainly of α-helices (43.23%±0.27%), whereas LDL was composed almost exclusively of intramolecular ß-sheets (67.36%±0.56%) or ß-turns, or both. On the other hand, whole egg yolk was mainly composed of intermolecular ß-sheets (39.77%±0.48%), potentially indicating molecular interchanges between the individual fractions.

Alkaline phosphatase in nasal secretion of cattle: biochemical and molecular characterisation.

BACKGROUND: Nasal secretion (NS) was investigated as a source of information regarding the mucosal and systemic immune status of cattle challenged by respiratory disease. A method for the collection of substantial volumes (~12 ml) of NS from cattle was developed to establish a reference range of analytes that are present in the NS of healthy cattle. Biochemical profiles of NS from a group of 38 healthy Holstein-Friesian cows revealed high alkaline phosphatase (AP) activity of up to 2392 IU/L. The character and source of the high activity of AP in bovine NS was investigated. RESULTS: Histochemical analysis confirmed the localization of the AP enzyme activity to epithelial cells and serous glands of the nasal respiratory mucosa. Analysis of mRNA levels from nasal mucosa by end point RT-PCR and PCR product sequencing confirmed that the AP was locally produced and is identical at the nucleotide level to the non-specific AP splice variant found in bovine liver, bone and kidney. Analysis by isoelectric focussing confirmed that AP was produced locally at a high level in nasal epithelium demonstrating that AP from nasal secretion and nasal mucosa had similar pI bands, though differing from those of the liver, kidney, bone and intestine, suggesting different post-translational modification (PTM) of AP in these tissues. CONCLUSIONS: A nasal isozyme of AP has been identified that is present at a high activity in NS, resulting from local production and showing distinctive PTM and may be active in NS as an anti-endotoxin mediator.

Proteomic analysis of intestinal mucosa responses to Salmonella enterica serovar typhimurium in naturally infected pig.

Salmonella enterica serovar typhimurium (S. typhimurium) is one of the most frequent Salmonella serotypes isolated from European pigs. Despite the advances in understanding the mechanisms involved in host-pathogen interactions and host cell responses to S. typhimurium, the global change that occurs in naturally exposed populations has been poorly characterized. Here, we present a proteomics study on intestinal mucosa of pigs naturally infected with S. typhimurium, in order to better understand the pathogenesis of salmonellosis and the pathways which might be affected after infection. Samples were analyzed by 2D-DIGE and 44 different proteins exhibited statistically significant differences. The data set was analyzed by employing the Ingenuity Pathway Analysis and the physiological function most significantly perturbed were immunological and infectious disease, cellular assembly and organization and metabolism. The pathways implicated in the porcine immune response to S. typhimurium were gluconeogenesis and Rho GDI/RhoA signaling, and our results suggest that keratins and the intermediate filaments could play an important role in the damage of the mucosa and in the success of infection. The role of these findings in salmonellosis has been discussed, as well as the importance of analyzing naturally infected animals to have a complete picture of the infection. Also, we compared the results found in this work with those obtained in a similar study using experimentally infected animals.

Liver proteomic analysis of the large yellow croaker (Pseudosciaena crocea) following polyriboinosinic:polyribocytidylic acid induction.

In the present study, we examined the liver protein profiles of the large yellow croaker (Pseudosciaena crocea) exposed to polyriboinosinic:polyribocytidylic acid [poly(I:C)], a viral mimic, using the differential proteomic approach. Sixteen altered protein spots were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry or matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry, including eight upregulated proteins and eight downregulated proteins. These altered host proteins were classified into six categories based on their biological function: cellular process, metabolic process, biological regulation, binding, and catabolic process, highlighting the fact that response to poly(I:C) induction in fish seems to be complex and diverse. Moreover, four corresponding genes of the differentially expressed proteins were validated by relative quantitative real-time PCR. Western blot analysis further demonstrated the changes in protein abundance of natural killer enhancing factor and peroxiredoxin 6. These results will be helpful in furthering our understanding of the changes of physiological processes in liver of fish during virus infection.

Serum amyloid A isoforms in serum and milk from cows with Staphylococcus aureus subclinical mastitis.

Serum amyloid A proteins (SAA) are very sensitive acute phase proteins, displaying multiple isoforms in plasma and different body fluids. They are currently under investigation as biomarkers of diseases. The aim of the present study was to compare the concentration and isoform expression of SAA in serum and milk of cows with bacteriologically negative milk (control group) and naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis (subclinical mastitis group). Somatic cell count (SCC) and bacteriological analyses were performed to establish the control and subclinical mastitis group. SAA concentration was evaluated using a commercial ELISA kit, while expression of different isoforms (serum A-SAA and milk M-SAA3 isoforms) was visualized by denaturing isoelectrical focusing and immunoblotting. The SAA concentrations in sera and milk of cows in the subclinical mastitis group were three and 100 times higher than in those from the control group of cows, respectively. Cows in the subclinical mastitis group had more acidic SAA isoforms in serum with the most prominent one at pI 5.5. This isoform was not detected in sera from the control group. Milk samples in the subclinical mastitis group contained abundant highly alkaline M-SAA3 isoforms and most of the serum isoforms, except for that at pI 5.5. In the subclinical mastitis group SAA isoforms with equivalent pI as serum isoforms accounted for 20% of the total SAA concentration in milk. There were significant differences in the concentrations and isoform patterns of SAA in serum and milk between the control and subclinical mastitis groups of cows. Also, we demonstrated that serum SAA isoforms were not transferred to milk proportion to their plasma content.

Serum amyloid A3 (SAA3), not SAA1 appears to be the major acute phase SAA isoform in the pig.

The acute-phase serum amyloid A (SAA) protein family comprises two main circulating (systemic) isoforms, SAA1 and SAA2, synthesised in liver and one local isoform, SAA3, produced in extrahepatic tissues. Systemic and local SAA show structural differences, which suggests different functions. In the pig, AA-amyloidosis is extremely uncommon, and the structural protein in swine has characteristics of systemic SAA. The only pig SAA sequences published so far, either derived form hepatic or extrahepatic sites have been designated SAA2, but the translated protein shows the properties of SAA3 proteins. The aim of this study was to characterise all the porcine SAA isoforms by sequencing from cDNA and genomic DNA obtained form multiple porcine tissues. Primer pairs were designed to amplify presumably all isoforms of SAA firstly and then specifically for each isotype. Results show that the only isotype isolated and sequenced both from hepatic and extrahepatic tissues correspond to a SAA3-like amino acid sequence. No SAA1-like sequences were identified, which could be indicative of the gene being very rare and consistent with the observed resistance to AA-amyloidosis. Finally, it is concluded that the pig is unique among other species in that the main circulating hepatic SAA isotype shows the characteristics of local highly alkaline SAA. This likely precludes a function as apolipoprotein.

Detection of oligoclonal bands in cerebrospinal fluid from German Shepherd dogs with degenerative myelopathy by isoelectric focusing and immunofixation.

BACKGROUND: Detection of intrathecal IgG synthesis is important in evaluating inflammatory diseases in the central nervous system. Isoelectric focusing (IEF) is currently the most sensitive method to demonstrate intrathecal IgG synthesis and may have diagnostic value for German Shepherd degenerative myelopathy (GSDM). OBJECTIVE: The objective of this study was to adapt a modified IEF and immunofixation method for the detection of oligoclonal bands in cerebrospinal fluid (CSF) from dogs with GSDM. METHODS: Serum and lumbar CSF samples were collected from 6 German Shepherd dogs clinically diagnosed with GSDM. Samples were also collected from 6 clinically healthy dogs for comparison. The concentration of IgG was measured by quantitative ELISA and the concentration of total protein was measured by the Bradford protein assay. The presence of oligoclonal bands was evaluated by use of modified IEF followed by immunofixation. RESULTS: The concentrations of total protein and IgG, and the IgG/total protein ratio in CSF samples, were not significantly different between GSDM patients and control dogs. Four GSDM patients had oligoclonal bands in their CSF based on IEF-immunofixation. No oligoclonal bands were found in CSF from control dogs. CONCLUSION: The results suggest that the detection of oligoclonal bands by IEF-immunofixation may have diagnostic value for GSDM, and support the idea that humoral immune responses may play a role in the pathogenesis of GSDM.

Proteomic profiling of Rhipicephalus (Boophilus) microplus midgut responses to infection with Babesia bovis.

Differences in protein expression in midgut tissue of uninfected and Babesia bovis-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus, were investigated in an effort to establish a proteome database containing proteins involved in successful pathogen transmission. The electrophoretic separation of midgut membrane proteins was greatly improved by using liquid-phase isoelectric focusing combined with one-dimensional or two-dimensional (2-D) gel electrophoresis. A selection of differentially expressed proteins were subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the identified Babesia-affected tick midgut proteins were six proteins that are implicated in signaling processes, including three Ca(2+)-binding proteins, a guanine nucleotide-binding protein, a protein with signal peptide activity and a translocon-associated receptor protein. Up-regulation of five metabolic enzymes indicated parasite-induced changes in electron and proton transport, protein processing and retinoic acid metabolism. Among the down-regulated proteins were a molecular chaperone, a cytoskeletal protein and a multifunctional protein of the prohibitin family. Identification of these proteins may provide new insights into the molecular interactions between B. bovis and its tick vector, and could lead to identification of anti-tick and transmission-blocking vaccine candidates.

Technical note: simultaneous identification of CSN1S2 A, B, C, and E alleles in goats by polymerase chain reaction-single strand conformation polymorphism.

Most variability in goat caseins originates from the high number of genetic polymorphisms often affecting the specific protein expression, with strong effects on milk composition traits and technological properties. At least 7 alleles have been found in the goat alpha(S2)-CN gene (CSN1S2). Five of them (CSN1S2*A, CSN1S2*B, CSN1S2*C, CSN1S2*E, and CSN1S2*F) are widespread in most breeds, whereas the other 2 (CSN1S2*D and CSN1S2*0) are rarer alleles. Four different PCR-RFLP tests are needed to detect all of these variants at the DNA level. The objective of this study was to develop and validate a rapid method for typing 4 of the 5 most-common goat CSN1S2 alleles by means of PCR-single strand conformation polymorphism (SSCP). The method was validated by analyzing 37 goat samples at the protein and DNA level, respectively, by milk isoelectrofocusing and PCR-RFLP methods already described. The genotypes obtained using the PCR-SSCP approach were in full agreement with those obtained by the validation analyses. The newly developed PCR-SSCP approach provides an accurate and inexpensive assay highly suitable for genotyping goat CSN1S2.

Short communication: Carora cattle show high variability in alpha(s1)-casein.

The objective of this study was to analyze the genetic variability of milk proteins of the Carora, a shorthorned Bos taurus cattle breed in Venezuela and in other Southern American countries that is primarily used for milk production. A total of 184 individual milk samples were collected from Carora cattle in 5 herds in Venezuela. The milk protein genes alpha(s1)-casein (CN) (CSN1S1), beta-CN (CSN2), kappa-CN (CSN3), and beta-lactoglobulin (LGB) were typed at the protein level by isoelectrofocusing. It was necessary to further analyze CSN1S1 at the DNA level by a PCR-based method to distinguish CSN1S1*G from B. Increased variation was found in particular at the CSN1S1 gene, where 4 variants were identified. The predominant variant was CSN1S1*B (frequency = 0.8). The second most common CSN1S1 variant was CSN1S1*G (0.101), followed by CSN1S1*C (0.082). Moreover, a new isoelectrofocusing pattern was identified, which may result from a novel CSN1S1 variant, named CSN1S1*I, migrating at an intermediate position between CSN1S1*B and CSN1S1*C. Six cows carried the variant at the heterozygous condition. For the other loci, predominance of CSN2*A2 (0.764), CSN3*B (0.609), and LGB*B (0.592) was observed. Haplotype frequencies (AF) at the CSN1S1-CSN2-CSN3 complex were also estimated by taking association into account. Only 7 haplotypes showed AF values >0.05, accounting for a cumulative frequency of 0.944. The predominant haplotype was B-A2-B (frequency = 0.418), followed by B-A2-A (0.213). The occurrence of the G variant is at a rather high frequency, which is of interest for selection within the Carora breed because of the negative association of this variant with the synthesis of the specific protein. From a cheese-making point of view, this variant is associated with improved milk-clotting parameters but is negatively associated with cheese ripening. Thus, milk protein typing should be routinely carried out in the breed, with particular emphasis on using a DNA test to detect the CSN1S*G variant. The CSN1S*G allele is likely to have descended from the Brown Swiss, which contributed to the Carora breed and also carries this allele.

Temporal changes in serum concentrations of acute phase proteins in newborn dairy calves.

Age-dependent changes in blood concentrations of four bovine acute phase proteins (APPs), serum amyloid A (SAA), lipopolysaccharide binding protein (LBP), haptoglobin (Hp) and alpha(1)-acid glycoprotein (AGP), were examined using two groups of newborn dairy calves. APP concentrations were monitored for either 3 weeks (Group A, n=13) or 2 months (Group B, n=13) after birth. Blood was collected at day 0-1 (Group A only), day 3 and then once or twice weekly until the end of the study. The possible transfer of colostral SAA as a source of SAA in the offspring was investigated by determining SAA isoforms in the serum of calves and in colostrum of their dams. Serum concentrations of all four APPs were high after birth, and concentrations showed a gradual decrease during the first 3 weeks of life. The lowest concentrations were at 21 days of age, after which concentrations stabilized. The calves synthesized adult SAA isotypes, and circulating SAA was not derived from colostrum. The results indicated that post-partum elevation of APPs is associated with the birth process and/or factors in colostrum and not necessarily with disease-related processes. This stresses the importance of considering a calf's age when interpreting APP concentrations in serum.

Effect of kappa-casein polymorphism on milk composition in the Orobica goat.

The aim of this work was to study the effects of isoelectrofocusing (IEF) milk protein variants on milk composition in the Italian Orobica goat breed, which is characterized by a rather high frequency of the kappa-casein (CSN3) B(IEF) allele. Significant associations were found between the IEF phenotype and protein and casein percentages. A favorable effect of the CSN3 B(IEF) variant was found for both protein and casein percentages, with a codominance trend for the 3 phenotypes: BB > AB > AA. Depending on the selection purpose, emphasis could be given to different kappa-casein variants in breeding. The high frequency of B(IEF) could be exploited in breeding strategies to improve the protein and casein percentages when cheese making is a selection objective.

Partial purification and characterization of Ascaridia galli diagnostic worm antigen.

Partial purification of Ascaridia galli whole worm extract was conducted by Cyanogen bromide Sepharose 4B immunoaffinity column chromatography. The resulted fraction was characterized by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The fraction was found to be consisted of six bands of 207 KDa, 157 KDa. 110 KDa, 103 KDa, 76 KDa and 41 KDa. This profile was compared with that of whole worm and excretory-secretory antigens. Both antigens were resolved into multiple bands in both high and low molecular weight ranges. The isoelectric focusing of the fraction displayed 8 bands of isoelectric points 7.5, 7.0, 6.8, 6.5, 6.2, 5.8. 5.3 and 4.6. The potency of this fraction in the diagnosis of natural ascaridiosis in chickens was assessed by ELISA compared with that of whole worm and ES antigens. The affinity purified fraction showed higher potentials in the diagnosis of A. galli infection in chickens than whole worm antigen at any sera dilution and than ES antigen at high sera dilutions. While ES antigen of the worms revealed higher diagnostic capabilities than whole worm extract. The current research recommends utilization of the affinity isolated fraction in the diagnosis of natural ascaridiosis in chickens.

First record of Trichinella pseudospiralis in the Slovak Republic found in domestic focus.

Infection of Trichinella spp. is widespread among wildlife in Slovakia and the red fox (Vulpes vulpes) is the main reservoir of Trichinella britovi. Trichinella spiralis has been rarely documented in sylvatic and domestic animals of this country. During routine examination of domestic pigs at the slaughter, Trichinella larvae were detected by artificial digestion in a domestic pig of a large-scale breeding farm in Eastern Slovakia. The parasite has been identified by molecular (PCR) and biochemical (allozymes) analyses and by the morphology of the nurse cell as the non-encapsulated species Trichinella pseudospiralis infecting both mammals and birds. The epidemiological investigation carried out at the farm level revealed the presence of the same parasite species in other three pigs of 192 examined (2.1%), in 3 of 14 (21.4%) examined synanthropic rats (Rattus norvegicus) and in a domestic cat. The farm was characterized by inadequate sanitary conditions, insufficient nutrition, cannibalism and the presence of rat population. A different profile has been observed at the phosphoglucomutase locus in T. pseudospiralis isolates from Slovakia in comparison with the T. pseudospiralis reference isolate from the Palearctic region. This is the first documented focus of T. pseudospiralis from Central Europe. The detection in domestic pigs of a non-encapsulated parasite infecting both mammals and birds stresses the need to avoid the use of trichinelloscopy to detect this infection at the slaughterhouse.

Kinetics of local and systemic isoforms of serum amyloid A in bovine mastitic milk.

The aim of the present study was to characterise the serum amyloid A (SAA) response to intramammary inoculation of Escherichia coli and to examine the distribution of hepatically and extrahepatically produced SAA isoforms in plasma and milk from cows with mastitis. Milk and plasma SAA concentrations were determined before and after experimental induction of E. coli mastitis in six dairy cows. The milk SAA response was characterised by low or undetectable levels before inoculation, very rapid and large increases in concentration after inoculation, and rapid decline towards baseline levels after resolution of disease. In plasma from cows with experimentally induced E. coli mastitis, four hepatically derived SAA isoforms with apparent isoelectric point (pI) values of 5.8, 6.2, 6.8 and 7.4 were demonstrated by denaturing isoelectric focusing. In milk three highly alkaline isoforms with apparent pI values above 9.3 appeared 12 h post-inoculation. These isoforms were not present in any of the plasma samples, and it therefore seems likely that they were locally produced, tissue-specific isoforms. At 24-36 h post-inoculation one or more acidic isoforms corresponding to those found in plasma appeared in the milk samples. The isoforms demonstrated in plasma from cows with E. coli mastitis were also present in serum obtained from three cows with clinical Streptococcus uberis mastitis. In conclusion, experimentally induced E. coli mastitis is accompanied by a prominent SAA response. The results of the present study indicate that SAA accumulation in mastitic milk is the result of both local synthesis of SAA and of hepatically derived SAA gaining access to the milk due to increased permeability of the blood-milk barrier.

Genetic relationships between sheep, cattle and human Echinococcus infection in Tunisia.

Allozyme variation at seven polymorphic loci (GPI, EST, MDH, MPI, DIA, PEP, PGM) was studied to examine genetic variation within and between sheep, cattle and human populations of Echinococcus granulosus in Tunisia. A high degree of genetic similarity was shown between the cysts of the three host origins. Nevertheless, whereas, the ovine and human samples were highly similar, the cattle samples were slightly different genetically. We conclude that humans are mostly infected by parasites originating from sheep liver. The intense deficiency in heterozygotes was partly artefactual (Wahlund effect) and partly due to self-fertilisation.

Short communication: Simultaneous identification of five kappa-casein (CSN3) alleles in domestic goat by polymerase chain reaction-single strand conformation polymorphism.

Until now, a total of nine polymorphic sites corresponding to six different alleles have been described at the kappa-casein (CSN3) locus in the domestic goat (Capra hircus). A protocol for the rapid and simultaneous genotyping of five goat CSN3 alleles by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique was developed. Moreover, the developed test was validated by screening the CSN3 variability in four Italian breeds, Garganica, Jonica, Maltese, and Camosciata. Seven different patterns were readily identifiable. These corresponded to five known alleles and two newly identified variants. The G/A substitution at nucleotide position 471, which is not identifiable at the protein level but was found to be very frequent in the typed breeds, is easily detectable by the protocol developed. The PCR-SSCP analysis is a powerful tool for the genetic study of CSN3 variability in domestic goats, allowing both the simultaneous identification of different alleles, and the detection of new variants.