Development and characterization of mouse monoclonal antibodies reactive with chicken IL-1ß.

Interleukin-1ß proteins from chicken, duck, goose, turkey, and pigeon share 77 to 99% amino acid sequence similarity among themselves, and only 31 to 35% sequence similarity is shared between avian and mammalian IL-1ß. There have been no antibodies that specifically detect avian IL-1ß, and the current study was conducted to develop mouse monoclonal antibodies (mAb) against chicken IL-1ß (chIL-1ß) to further define its biochemical and immunological properties. In this study, 2 mouse mAb that are specific for chIL-1ß were produced and characterized. Both mAb identified a 66.0 kDa recombinant chIL-1ß protein expressed in Escherichia coli by Western blot analysis that corresponded to the expected molecular weight of a recombinant fusion protein containing the full-length 23.0 kDa chIL-1ß protein and a 43.0 kDa maltose binding protein tag. Immunohistochemical analysis identified cells producing endogenous chIL-1ß in the cecal tonsils, bursa of Fabricius, and spleen. Purified recombinant chIL-1ß dose-dependently stimulated the proliferation and nitric oxide production by thymocytes, and both activities were inhibited by co-incubation with the 2 chIL-1ß mAb described in this paper. These mAb will be important immune reagents for basic and applied poultry research of IL-1ß in poultry.

[Analysis of the duration of and reasons for changing the first combination of antiretroviral therapy]. / Análisis de la duración y los motivos de cambio de la primera combinación de tratamiento antirretroviral.

OBJECTIVE: To determine the duration of and reasons behind changing the various combinations of drugs used for the initiation of antiretroviral treatment in naïve patients. METHODS: A retrospective observational study that included all patients with HIV infection who started antiretroviral therapy in a high-tech university reference hospital during the period from 1 January 2003 and 31 December 2005. Patients were followed until 31 December 2008. To estimate the cumulative probability of discontinuation the Kaplan-Meier method was used. RESULTS: A total of 441 patients were included. The average duration of the first treatment was 384 (interquartile interval 84-1290) days. The regimen based on non-nucleoside reverse transcriptase inhibitors and those that included as nucleosides abacavir or tenofovir in combination with lamivudine or emtricitabine showed a significantly longer duration than the rest. The main reasons for termination were the side effects, although in a lesser percentage than that obtained in previous studies. No associations were found between the rest of the characteristics of the patients or of the treatment and the risk of termination. DISCUSSION: Although the duration of the first antiretroviral treatment remains short, currently fewer changes are made due to side effects and due to loss to follow-up. The reasons may be better tolerance and less complexity. However, more studies are needed to determine the benefits of one regimen or another, and to be able to generalise the results.

Pathogenesis of Eimeria praecox in chickens: virulence of field strains compared with laboratory strains of E. praecox and Eimeria acervulina.

The pathogenesis in chickens of the apicomplexan Eimeria praecox was compared with that of Eimeria acervulina, using intestinal lesions, mucosal integrity, body weight gain (BWG) and the feed conversion ratio (FCR) as criteria. Characteristics of each species were described by combinations of polymerase chain reaction assays and classic parasitological signs. There were considerable overlaps in lengths, breadths, shape indices and volumes of the oocysts of each species. Both species caused statistically significant reductions in BWG at the lowest inocula tested (500,000 sporulated oocysts per bird of E. praecox and 250,000 of E. acervulina). E. praecox was observed for the first time to cause actual body weight loss and marked increases in FCR, as did E. acervulina. E. acervulina caused gross, white pathognomonic lesions, but E. praecox caused micro-lesions, visible in fresh tissue only with a dissecting microscope. Occasionally, lesions of the Houghton strain of E. acervulina were observed to be rounded, rather than typically "ladder-like". Both species caused villous erosion and atrophy. No mortality occurred in birds receiving up to 1 million sporulated oocysts of either species. Using BWG and FCR as criteria, the virulence of recent field strains of E. praecox from Wales (Tynygongl) and the USA (Raleigh) was compared with English laboratory strains of E. praecox (Houghton) and E. acervulina (Houghton). E. praecox (Tynygongl) was markedly more virulent than E. acervulina (Houghton), which was more virulent than E. praecox (Raleigh) and E. praecox (Houghton).

Cryptosporidium species and subtype analysis from dairy calves in Spain.

Faecal specimens from 287 diarrhoeic calves younger than 21 days, collected over a 2-year period (2006-2007) from 82 dairy cattle farms in 14 provinces across the north of Spain, were examined for the presence of Cryptosporidium oocysts. Overall, 63 farms (76.8%) and 166 calves (57.8%) tested positive by microscopy. In order to elucidate the genetic diversity, selected positive specimens from 149 calves originating from 61 farms in the 14 provinces were examined by genotyping and subtyping techniques. Cryptosporidium parvum was the only species identified by PCR-RFLP of SSU rDNA from all 149 isolates and sequencing of a subset of 50 isolates, except for 2 specimens that were identified as C. bovis. Sequence analyses of the glycoprotein (GP60) gene revealed that most C. parvum isolates (98%) belonged to the subtype family IIa and 2 isolates were identified as the novel subtype IIdA23G1. Subtype IIaA15G2R1 was the most common and widely distributed (80.3% of the 61 farms), followed by subtype IIaA16G3R1 (14.7%), whereas the remaining IIa subtypes (IIaA16G2R1, IIaA17G2R1, IIaA18G3R1, IIaA19G3R1) were restricted to 1-3 farms. All these C. parvum IIa subtypes have previously been described in human patients, indicating that most isolates from diarrhoeic calves in northern Spain have zoonotic potential.

Effect of the quinolone coccidiostat decoquinate on the rearrangement of chromosomes of Eimeria tenella.

The present report concerns our attempts to further study the effect of quinolone coccidiostats on the sporulation of Eimeria tenella oocysts by analyzing the meiotic behaviour of the chromosomes. To that end, synaptonemal complexes were analyzed by TEM applied to intact meiotic chromosomes. These were isolated after disruption of oocysts, which were harvested from decoquinate-medicated and non-medicated (control) birds. In oocysts from control birds, synaptonemal complexes appeared as the 14 bivalents of the normal karyotype. However, in oocysts from medicated birds, our synaptonemal complex analysis revealed a reciprocal translocation, which was observed as an irregular pairing of chromosome axes 5 and 12 resulting in quadrivalent and trivalent configurations. This finding suggests breakage points in chromosomes 5 and 12 and exchange of chromosomal segments. Furthermore, breakpoints in chromosome 12 resulted in telomere deletion. The chromosomal aberrations described in the present study may result in reduced sporulation since chromosomes involved in translocations segregate abnormally during meiosis. In addition, the results reported provide new evidence of the inhibitory effect of quinolones on the sporulation of E. tenella oocysts, since sporocysts were not formed.

Differences in Hsp70 expression in the sporozoites of the original strain and precocious lines of Eimeria tenella.

The levels of expression of Heat shock protein 70 (Hsp 70) in sporozoites of a wild-type parent strain and 2 precocious lines of Eimeria tenella, were compared to investigate the relationship between the heat shock proteins expressed by the parasite and virulence of the strain. Hsp70 expression was analyzed in sporozoites by immunohistochemical techniques, immunoblot, and flow cytometric analyses. One band of 70 kDa was identified and the variation of the Hsp70 expression levels was quantified by optical densitometric analyses. The results showed a significant gradual decrease in the Hsp70 expression in sporozoites of E. tenella as attenuation progressed, suggesting that the Hsp70 expressed in the excysted sporozoites of E. tenella might be involved in parasite pathogenicity. In addition, the cytoplasmic distribution of the Hsp70, which was observed in the entire sporozoites of the wild strain, was reduced to the anterior portion in the precocious lines.

Ultrastructural localization of a soluble antigen in the chicken Harderian gland.

The relationship between plasma cells, macrophages, B and T cells, dendritic cells, and epithelium in the chicken Harderian gland have been studied by means of ultrastructural localization of the horseradish peroxidase following local immunization. After 5 d, peroxidase activity was found in vesicles located in macrophages and immature plasma cells. On day 7, peroxidase-antiperoxidase complexes were found in vesicles of the epithelial cells lining the secondary ducts and the acini, in the lumina of the ducts, and on the surface of lymphocytes located among these epithelial cells. Dendritic cells showing peroxidase activity on their surface were seen in the subepithelial lymphoid tissue and in the lymphoid follicles. On day 9, peroxidase activity was found as iccosomes on the surface of dendritic cells and lymphoblasts. These results indicate that immature plasma cells in the Harderian gland can take up antigen and may have a role in presenting it to T cells. Further, our results suggest that intraepithelial lymphocytes might be involved in antigen transportation from the epithelium to the subepithelial lymphoid tissue.

Anti-S-100 antibody recognizes ellipsoid-associated cells and other dendritic cells in the chicken spleen.

The chicken spleen was studied immunohistochemically with anti-S-100 protein polyclonal antibody. S-100-positive cells accumulated around the penicilliform capillaries during the first 3 weeks of life. After 2 weeks posthatch the S-100-positive cells appeared in the red pulp, periarterial lymphatic sheath, and subsequently in the germinal center. Their ontogenetic development and intrasplenic distribution strongly suggested that the S-100-positive cells were identical with ellipsoid-associated cells. The S-100-negative cells of the periellipsoidal white pulp gradually transformed to S-100-positive, functionally active cells on the surface of the ellipsoid. The immunohistological findings support the hypothesis that the interdigitating dendritic cells and follicular dendritic cells were not of monocytic origin but belong to a splenic resident, endocytic cell line located on the surface of the ellipsoid.

Eimeria necatrix virus: intracellular localisation of viral particles and proteins.

The presence of the Eimeria necatrix virus was investigated in the following life cycle stages: sporocysts, sporozoites, merozoites, and macrogametes. Electron microscopy revealed virus-like particles (VLPs) in sporozoites, which were purified from sporozoite extracts and used to raise polyclonal antibodies. Viral proteins were identified as RNA polymerase (95 kDa) and the major capsid protein (80 kDa). Polyclonal antibody was used to detect the intracellular localisation of VLPs and proteins. Immunoelectron microscopy and immunohistochemistry identified a viral protein of 95 kDa in all the E. necatrix stages studied, whereas the 80 kDa protein was found only in sporocysts and sporozoites. In addition, no VLPs were found in sporocysts. These results indicate that the synthesis of viral capsid proteins takes place during the early events of sporulation, and is then packaged into novel viruses during the late events. No VLPs were seen and no capsid proteins were found in the merozoites and macrogametes, whereas the 95 kDa RNA polymerase was present in both these stages. In addition, no VLPs or proteins were detected in chicken tissues.

Myofibroblasts and myoepithelial cells in the chicken harderian gland.

An electron microscopic study of the myoepithelial cells in the chicken Harderian gland provides evidence that these cells can be transformed into myofibroblasts. After the application of a Brucella ovis suspension in sterile saline onto the eyeball, every 5 minutes for half an hour, myoepithelial cells gradually develop over a 90-minute period the characteristic features of myofibroblasts: bundles of intracytoplasmic microfilament; abundant rough endoplasmic reticulum; prominent Golgi complex; and surface membrane differentiations, that provide attachment to neighbouring epithelial cells. No typical desmosomes are observed. Besides, the intercellular space between epithelial cells and myofibroblasts increases and the basement membrane adjacent to myofibroblasts disappears. Hypoxia is hypothesized to be involved in the transformation of myoepithelial cells into myofibroblasts.

Immune complex-mediated glomerulopathy in Barbus graellsi infected with Myxobolus spp.

Membranous glomerulonephritis caused in Barbus graellsi by myxosporidian infections have been studied by electron microscopy and immunoelectron microscopy techniques. This study indicates that Myxosporidian infection produces a chronic severe aggression. Spores reach the spleen, the kidney and the liver, where they are trapped and phagocyted by Melano Macrophage Centres. Consequently, the commencement of a immunological response to myxosporidian is evident. Our results show the presence of immunodeposits in the basement membrane of the glomeruli, suggesting that they might initiate glomerulonephritis. The lesion was markedly similar to immune complex-mediated glomerulonephritis disease in higher vertebrates.

Follicular dendritic cell activation in the harderian gland of the chicken.

The avian follicular dendritic cell changes that occur in the germinal center of the Harderian gland during the course of the immune response were studied by electron microscopy and the immunoperoxidase method was employed for the detection of S-100 protein. The chickens were injected twice with Salmonella O Antigen into the nictitating membrane at 9-day intervals. The follicular dendritic cells exhibited filiform processes at between 24 and 96 h after the second antigen administration. Filiform dendrites tended to convolute near the cell body. Therefore, it can be assumed that these processes make it more difficult for macrophages and B cells to make contact with the immune complexes retained by the follicular dendritic cells and, as a consequence, the period of antigen handling by these cells increases. Evidence is provided that the dendritic processes are closely associated with both lymphoblasts and lymphocytes. Furthermore, S-100 protein was found in the abovementioned cells exclusively and only in those cells where filiform dendrites were observed. These findings suggest that, during a secondary immune response, the follicular dendritic cell undergoes a functional activation which involves morphological changes and the phenotypic expression of the S-100 protein. This activation is hypothesized to be similar to that described for follicular dendritic cells in mammals after fixing immune complex.

Immunocytochemical and immunoelectron microscopy study of dendritic cells in the caecal tonsils of chickens infected with Eimeria tenella.

In the present study S-100 protein containing cells in the caecal tonsil were investigated, both at light microscopic and at electron microscopic levels, after oral coccidia inoculation (Eimeria tenella). The birds were infected with a single (Day 0) or two (Days 0 and 21) infective doses of 500 oocysts. Immunoelectron reactivity for S-100 protein was demonstrated in infected chickens, but not in controls. It was found in follicular dendritic cells and interdigitating dendritic cells which were present in the germinal center and diffuse lymphoid tissue respectively, both of them being located in the deep lymphoid tissue near the muscular layer and around the deep glands. Outside the lymphoid tissue, immunoreactivity for S-100 protein was found in cells lying between the epithelial cells of the deep crypt epithelium. Positivity for S-100 protein was observed at 3, 12, 18, 24 and 48 h after the first inoculation as well as on Days 3, 5, 6, 7, 8, 25 and 30 of the experiment. Positive reaction for S-100 protein was detected both while schizont (the more immunogenic stage) development occurs and the number of sporozoites in the caeca lumen was higher, as well as when the production of oocysts reached a maximum. Complementary studies demonstrated that S-100 positive dendritic cells gave a negative reaction for esterase activity, whereas a subset of S-100 negative intraepithelial lymphocytes located between epithelial cells lining the deep glands exhibited esterase activity. These esterase positive cells are hypothesized to be involved in the regulation of local defences.

Cytochemical study of the germinal membrane of the Echinococcus granulosus cyst.

The present cytochemical study was undertaken to provide more information on the localization of enzymatic and glycoconjugates in the germinal membrane of the Echinococcus granulosus cyst. The distinctive distribution of binding sites for two lectins (peanut agglutinin and Dilochos biflorus agglutinin) in the germinal membrane are described. An investigation is made of the distribution and specific activity of adenosine triphosphatase, alkaline phosphatase and acid phosphatase. The results suggest that cells located in the deeper layer of the germinal membrane are intrinsic in the cellular differentiation process. The dissimilarities detected in both the enzymatic activity and the lectin-binding receptors could be associated with metacestode development or degeneration.

Prevalence of intestinal parasites, including Cryptosporidium parvum, in dogs in Zaragoza city, Spain.

Faecal samples from 81 dogs aged between 2 months and 13 years were collected in the small animal clinic (37 domestic dogs) and the animal shelter (44 stray dogs) located in the Faculty of Veterinary Sciences in Zaragoza city (northeast Spain) and screened for the presence of Cryptosporidium oocysts. Faeces were concentrated by the formalin-ethyl acetate method and smears of the sediment were stained by using the modified Ziehl-Neelsen technique. Cryptosporidium parvum oocysts were detected in six dogs (7.4%) aged from 2 months to 6 years. Infection was detected in both domestic (three) and stray (three) dogs and all of them excreted few oocysts (0-1 oocyst per 20 x field). No statistically significant differences in prevalence occurred between dogs younger than 6 months (11.8%) and the older dogs (6.2%). Prevalences were not significantly different between domestic (8.1%) and stray dogs (6.8%). Diarrhoea was recorded in three of the positive dogs (50%), although additional enteric parasites such as oocysts of Isospora spp. were also detected in their faeces. Nevertheless, prevalence was significantly higher in diarrhoeic (30%) versus non-diarrhoeic (4.2%) dogs (P < 0.05). Cryptosporidium was one of the parasites most frequently detected in the dogs surveyed.

Immunohistochemical study of the cyst of Besnoitia besnoiti.

The present study has been undertaken in order to provide information on the molecular structure of the cysts of Besnoitia besnoiti. To that end, immunohistochemical techniques have been used to investigate the expression of several enzymes and proteins implicated in the cellular membrane permeability of bradyzoites. Paraffin and frozen sections, which were obtained from subcutaneous tissue samples taken from naturally infected cattle (coming from northeast Spain), were treated with a panel of antibodies. These were specific for Na(+), K(+)-ATPase, alkaline phosphatase, calmodulin, S100 protein, heat shock proteins, hsp60, and hsp70. Positive-cysts for the said antibodies were found in 23.3% of the cows studied. Bradyzoites showed a positive immunoreaction in every positive cyst with respect to all these antibodies. In addition to the low percentage of positive animals, it is worth noting that positive and unstained cysts were observed in the same tissue section. These results suggest that bradyzoites may pass through both active and dormant metabolic phases.

A method for the sequential study of eimerian chromosomes by light and electron microscopy.

In the present study, the authors describe a simple method to isolate chromosomes from eimerian oocysts and to submit them to sequential study by light and electron microscopy. This method includes a reliable and reproducible technique for transferring eimerian chromosomes from slides to grid that fulfills the essential requirements for generalized use in cytogenetics. In addition, this method overcomes the difficulty of the resistance of protozoan oocysts to disruption and permits the release of intact meiotic chromosomes. The observation by the authors of synaptonemal complexes in meiotic chromosomes of different Eimeria species by applying the above-mentioned method to oocysts revealed its importance to future applications.

Expression of anti-apoptotic factors in cells parasitized by second-generation schizonts of Eimeria tenella and Eimeria necatrix.

Intracellular infections by parasites require a functional anti-apoptotic mechanism for parasite survival within the host cell. The intracellular cycle of Eimeria tenella and Eimeria necatrix in chicken intestinal cells involves the maturation of schizonts within the epithelial cells lining the crypt lumen of the ceca (E. tenella) and jejunum (E. necatrix). After invasion, these cells detach from the epithelial layer and migrate into the underlying connective tissue, where maturation of second-generation schizonts takes place. However, the detached epithelial cells that harbour the parasite and localize in the lamina propia do not undergo apoptosis despite the fact that they are parasitized cells and are located in an inappropriate microenvironment. In this study we consider the hypothesis that E. tenella and E. necatrix may inhibit the host cell apoptosis that accompanies parasite-mediated transformation during late schizogony. To that end, the expression of both NF-kappaB, a transcriptional factor that blocks parasite-induced apoptosis, and bcl-xL, an anti-apoptotic protein induced by NF-kappaB, were studied in the host cell during the maturation of second-generation schizonts. In addition, the expression of the phosphorylated inhibitor of NF-kappaB, p-IkBalpha, was also studied to further confirm NF-kappaB activation. Immunocytochemical techniques, flow cytometric and blott analysis were applied by using polyclonal antibodies that specifically react with bcl-xL, p-IkBalpha, and NF-kappaB to detect these anti-apoptotic proteins in the parasitized cell. Our results offer evidence that both these coccidial species first induce NF-kappaB activation to protect the transformed parasitized cells from apoptosis, allowing the second-generation schizonts to mature, and later, after complete schizonts maturation, cause NF-kappaB inhibition to trigger host cell apoptosis in order to facilitate the escape of merozoites. To determine whether inhibition of the NF-kappaB pathway would induce apoptosis of the host cell, a protease inhibitor (TPCK), which induces apoptosis by mediating inhibition of IkB phosphorylation, was administered to parasitized chickens.

Identification of a fibronectin-like molecule on the surface of Leishmania amastigotes.

The major surface glycoprotein of Leishmania gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. In this article, we describe a cross-reactivity between an anti-fibronectin monoclonal antibody and the amastigote gp63 by means of immunoelectron microscopy and immunoblot. Immunoreactivity was found on the amastigote membrane and in the flagellar pocket. We suggest that gp63 may play a role in the protection and/or nutrition process of the parasite in the phagolysosomes of the macrophage.

Field trial on the therapeutic efficacy of paromomycin on natural Cryptosporidium parvum infections in lambs.

The objective of this study was to evaluate the therapeutic efficacy of paromomycin against cryptosporidiosis in naturally infected lambs under field conditions. The 36 cross-bred neonatal lambs, 3-10 days old, were used. On the first day that lambs showed diarrhea (Day 1) they were randomly divided into three groups. The infected control group (14 lambs) remained unmedicated whereas the two other groups were orally medicated with paromomycin solution (Humatin((R)), Parke Davis, France): 12 lambs (Group A) at 100mg/kg per day for three consecutive days (Days 1-3) and 10 lambs (Group B) at 200mg/kg per day for two days (Days 1 and 2). Drug efficacy was assessed by evaluating the presence of diarrhea, oocyst shedding and weight gains from Days 1 to 23. The results show the efficacy of paromomycin in reducing both cryptosporidial oocyst output and severity of clinical signs. On Day 4, all unmedicated lambs remained infected and excreted large numbers of cryptosporidial oocysts (mean score: 2.5) whereas oocyst output had stopped in most medicated lambs (>60%) and low numbers of oocysts were excreted in the remaining lambs (mean score: 0.45 in Group A and 1 in Group B). Mean oocyst excretion was significantly reduced in medicated lambs from Days 2 to 5 (P<0.05). Treatment also reduced, but not completely prevented, clinical symptoms although diarrhea stopped in most medicated lambs just after drug withdrawal. The mean weight gains of Group A lambs were higher than that of unmedicated lambs throughout the study and statistically significant differences were found from Days 1 to 11 (1.99+/-0.81 versus 1.47+/-0.53) (P<0.05). By contrast, the growth rate of Group B lambs from Days 11 to 23 was impaired when compared with the two other groups (P<0.05) although no significant differences were found at the end of the study (Days 1-23).