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.

Relationship between function, strength and electromyography of upper extremities of persons with tetraplegia.

STUDY DESIGN: Cross-sectional. OBJECTIVE: To analyze the relationships between functional tests, arm strength and root mean square of surface electromyography (EMG). SETTING: Sao Paulo, Brazil. METHODS: Twenty-four individuals with chronic tetraplegia participated. Upper extremity motor score (UEMS), functional independence measure (FIM) motor score, spinal cord independence measure III and capabilities of upper extremity (CUE) were performed. Muscle strength of the right elbow flexors-extensors was assessed using dynamometry and manual muscle test (MMT) and EMG of right biceps and triceps brachii were performed. Spearman's rank correlation coefficients and Mann-Whitney's U-test were used. RESULTS: Functional tests and UEMS correlated strongly among them. UEMS highly correlated with triceps dynamometry and EMG. The dynamometry showed a very high correlation with MMT on the extensor group and a moderate correlation with flexor group. Triceps EMG showed moderate correlation with FIM and CUE. High correlations between triceps EMG and elbow extensors dynamometry and MMT were observed. A significant better performance on functional tests was observed on lower ASIA motor levels. The low-tetraplegia group showed a significant higher score on triceps EMG and dynamometry. CONCLUSION: Arm strength and EMG seem to be related to functional capabilities and independence in chronic tetraplegia.

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

Objetivo Conocer la duración y los motivos de cambio de las distintas combinaciones de fármacos utilizadas como inicio del tratamiento antirretroviral en pacientes naive. Métodos Estudio observacional y retrospectivo en el que se incluyeron todos los pacientes con infección por VIH que iniciaron tratamiento antirretroviral en un hospital universitario de referencia de alta tecnología durante el periodo comprendido entre el 1 de enero de 2003 y el 31 de diciembre de 2005. El seguimiento se realizó hasta el 31 de diciembre de 2008. Para estimar la probabilidad acumulada de interrupción del tratamiento se utilizó el método de Kaplan-Meier. Resultados Se incluyeron un total de 441 pacientes. La mediana de duración del primer tratamiento fue de 384 (intervalo intercuartil 84–1.290) días. Los regímenes basados en inhibidores de la transcriptasa inversa no análogos de nucleósidos y aquellos que incluían como análogos de nucleósidos abacavir o tenofovir en combinación con lamivudina o emtricitabina presentaron una duración significativamente mayor que el resto. Los principales motivos de finalización fueron las reacciones adversas aunque en un porcentaje menor que el obtenido en estudios anteriores. No se hallaron asociaciones entre el resto de características de los pacientes o del tratamiento y el riesgo de interrupción. Discusión Aunque la duración del primer tratamiento antirretroviral sigue siendo corta, actualmente se producen menos cambios por reacciones adversas y por pérdidas de seguimiento. Los motivos podrían ser una mejor tolerancia y una menor complejidad. No obstante, son necesarios más estudios para determinar el beneficio de un régimen frente a otro y poder generalizar estos resultados (AU)

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 (AU)

[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.

Relationship between adherence level, type of the antiretroviral regimen, and plasma HIV type 1 RNA viral load: a prospective cohort study.

The relationship between adherence, antiretroviral regimen, and viral load (VL) suppression was assessed through a 1 year prospective follow-up study among 1142 HIV-infected patient. Patients on antiretroviral therapy who attended to the pharmacy during a 6-month period were considered eligible. Those included in the final analysis were patients who had been taking the same antiretroviral therapy for > or =6 months since their inclusion. The cohort included patients taking first line therapy (n = 243) and antiretroviral-experienced patients (n = 899). Naive patients who were included had to have reached undetectable VL at enrollment. Antiretroviral-experienced patients with detectable VL determinations in the previous 6 months were excluded. Adherence was measured by means of announced pill counts and dispensation pharmacy records. Of patients, 58% were taking NNRTI, 31.4% boosted PI, and 10.6% unboosted PI-based regimens. Overall, the relative risk of virologic failure was 9.0 (95% CI 4.0-20.1) in patients with adherence 80-89.9%, 45.6 (95% CI 19.9-104.5) with adherence 70-79.9%, and 77.3 (95% CI 34.2-174.9) with adherence <70%, compared with adherence of > or =90%. The risk of virologic failure in patients with adherence <90% taking unboosted PI was 2.5 times higher than the group taking boosted PI (95% CI 1.2-5.3). There were no statistical differences in patients taking boosted PI and those who were taking NNRTI. Less than 95% of adherence is associated with high virologic success. For patients taking NNRTI- or boosted PI-based regimens with adherence rates of 80%, the failure rate is <10%. These data do not affect the goal of achieving the highest level of adherence possible.

Mouse bioassay for palytoxin. Specific symptoms and dose-response against dose-death time relationships.

Nowadays, a variety of protocols are applied to quantitate palytoxin. However, there is not desirable agreement among them, the confidence intervals of the basic toxicological parameters are too wide and the formal descriptions lack the necessary generality to establish comparisons. Currently, the mouse bioassay is the most accepted one to categorize marine toxins and it must constitute the reference for other methods. In the present work, the mouse bioassay for palytoxin is deeply analyzed and carefully described showing the initial symptoms of injected mice which are presented here in the first time. These symptoms clearly differ from the more common marine toxins described up to now. Regarding to the toxicological aspects two considerations are taking into account: (i) the empiric models based in the dose-death time relationships cause serious ambiguities and (ii) the traditional moving average method contains in its regular use any inaccuracy elements. Herein is demonstrated that the logistic equation and the accumulative function of Weibull's distribution (with the modifications proposed) generate satisfactory toxicological descriptions in all the respects.

Calcineurin inhibitors affect circulating regulatory T cells in stable renal transplant recipients.

INTRODUCTION: Immunosuppression, although crucial for short-term management, has been described in renal transplantation to be a major hurdle for long-term graft survival. Efforts have been directed at achieving a true state of allotolerance, thereby reducing the load of immunosuppression. Recently, increased frequencies of CD4(+)CD25(high) regulatory T cells (Tregs) have been described as an additional mechanism to induce alloimmune tolerance. MATERIALS AND METHODS: We assessed 64 renal transplant recipients with stable renal function for at least 1 year, divided into two groups: one composed of patients receiving rapamycin but not calcineurin inhibitors (CNIs), and another, of those receiving CNIs but not rapamycin. RESULTS: We demonstrated that T cells with a regulatory phenotype were decreased in peripheral blood of renal transplant recipients under CNI therapy compared to those who were CNI-free. The Tregs in our patients showed a modest association with renal function as measured by the delta serum creatinine, which was not significant. CONCLUSIONS: CNIs, but not rapamycin, reduce the frequencies of circulating Tregs in renal transplant recipients. The use of rapamycin might be further exploited in strategies reducing immunosuppression in renal transplantation. Furthermore, quantification of blood Tregs may be a suitable tool to identify those recipients who are candidates for reducing immunosuppression.

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.

Estudo de confiabilidade da aplicação da escala de Fugl-Meyer no Brasil / Reliability study on the application of the Fugl-Meyer scale in Brazil

OBJETIVOS: Os objetivos do estudo foram realizar uma versão brasileira da escala original de Fugl-Meyer e verificar a confiabilidade da aplicação inter e intra-observador desta versão em pacientes crônicos pós AVC. MÉTODO: Participaram do estudo 50 pacientes portadores de hemiparesia, os quais foram submetidos a duas avaliações (confiabilidade intra-observador), realizadas por três fisioterapeutas (confiabilidade interobservador), procedentes de três centros de reabilitação. RESULTADOS: Os resultados demonstraram alta confiabilidade inter e intra-observador da EFM total (IC = 0,99 e 0,98; respectivamente), assim como para todas as subescalas (interobservador IC = 0,99 a 0,94; intra-observador IC = 0,98 a 0,87). CONCLUSÃO: Conclui-se neste artigo que não foi verificado conflitos de interpretação na versão brasileira da escala de Fugl-Meyer. Obtivemos alto índice de confiabilidade, tanto intra como interobservador, permitindo assim seu uso como instrumento de avaliação clínica e de pesquisa no Brasil.

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.

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.

[Resistance to antiretroviral therapy]. / Resistencias al tratamiento antirretroviral.

OVERVIEW: After some time under treatment, HIV+ patients have a virologic failure rate of 50%, being development of resistance to therapy responsible for up to 80% of the virologic failure. In addition, resistance rates in naive patients is around 10% in developed countries. Inherent characteristics of HIV (replication cycle, viral subtype), of patients (therapy compliance, intra-/interindividual variability, genetic polymorphisms), and of therapy (genetic barrier to drug resistance, inhibitory ratio, drug interactions) are the factors involved in the development of resistance, and their interpretation requires to be studied. Resistance identification will be carried out using genotypical and/or phenotypical methods, and their adequacy has been validated by various expert panels on resistance. The role of the pharmacokinetic and pharmacodynamic monitoring of antiretroviral therapy is also crucial within the field of resistance, and concerns us directly as pharmacists. Finally, understanding the resistance patterns of currently available or experimental antiretroviral drug families will provide the necessary tools to prevent and/or manage their development. OBJECTIVES: To know and understand the mechanisms and patterns of resistance for each antiretroviral family. To identify factors involved in the development of resistance to ART, and to interpret various resistance tests. SEARCH STRATEGY: Studies were identified using Medline, the Cochrane database of systemic reviews, abstracts from international meetings on AIDS, Conference on Retroviruses and Opportunistic Infections, international meetings on resistance to antiretrovirals, and product monographs from January 1999 to February 2004. SELECTION CRITERIA: To be eligible, studies had to describe viral genome mutations responsible for resistance or hypersusceptibility to ART in relation to precipitating factors. Papers describing resistance identification techniques were also selected. DATA COLLECTION AND ANALYSIS: In all, 1,083 full articles and 64 abstracts and communications at international meetings were retrieved, of which 74 articles and 20 abstracts met the inclusion criteria for our review. PRIMARY RESULTS: Of the 94 reports selected, 86 discussed factors involved in the development of resistance and resistance test interpretation. The remaining 8 reports focused on resistance patterns to the various antiretroviral drug families. Every article described the enzymatic mechanisms induced by mutations responsible for resistance or hypersusceptibility to each antiretroviral family, the classification and nomenclature for each mutation, and the influence of each mutation on the success or failure of patient treatment. REVIEWER S CONCLUSIONS: Knowledge of the mechanisms and patterns of resistance to each antiretroviral family will allow us to overall understand the evolution and outcome of treatment for any given patient. Only thus shall we be able to play an integral role in the therapy of patients.

Phenology of the tick, Ixodes ricinus, in its southern distribution range (central Spain).

The abundance, seasonal activity patterns and development rates of the tick Ixodes ricinus (L.) (Acari: Ixodidae), as well as microclimate features of the site of study, are described for a 9-year-long study (1994-2002) in north-central Spain. According to drag captures, larvae had a unimodal activity pattern, with a maximum observed around July-August, whereas nymphs displayed a bimodal pattern (May-June and August-September) with strong dominance of spring activity. An inversion of this pattern, with larger autumn peak, was observed in years with humid summers. Adults showed a small spring peak and a large autumn one. In the later years of the study, a small increase in the adult spring peak of activity was noticed, correlated with mild winters. Over the entire period of study, a clear increase in the total tick abundance was detected. Statistically significant differences between years were observed for some climate variables (saturation deficit, winter temperatures and number of days with temperatures above 6 degrees C), but a consistent and constant pattern of change was not observed in any climate variable. Temperature requirements for developing stages showed a sharp decrease in weeks 35-51 (eggs) and 38-50 (larvae and nymphs), a feature attributed to the presence of the morphogenetic diapause, beginning around September. Development rates obtained under quasi-natural conditions were almost twice those reported for other sites, suggesting an adaptation of this local, largely isolated I. ricinus population. According to drag captures and field-obtained development rates, interchange of nymphs between the two cohorts is common in this site, and seems to be influenced by the winter temperature and the date of larval engorgement.

Consumo patológico de meperidina en una paciente afecta de porfiria aguda intermitente / Pathological consumption of meperidi ne in a patient with acute intermittent porphyria

Objetivo: Presentación de un caso de consumo patológico de meperidina, así como de su tratamiento. Discusión acerca del término "pseudoadicción". Paciente: Paciente afectado de porfiria aguda intermitente, consumidora patológica de meperidina. Es ingresada para estudio del cuadro. Se inicia tratamiento de deshabituación. Resultados: La paciente fue tratada satisfactoriamente mediante tratamiento multidisciplinar con medicación psiquiátrica, metadona e ibuprofeno. La paciente no presentaba adicción a opiáceos según la definición estricta de consumo patológico no asociado a la búsqueda de analgesia. Cumplía los términos para diagnosticársele pseudoadicción. Discusión: Durante la práctica clínica, se pueden hallar casos de pacientes con dolor crónico con conductas patológicas de consumo de fármacos. Se puede prevenir mediante la adecuada analgesia o, una vez iniciado el cuadro, corrigiendo los mecanismos de perpetuación. (AU)

Prevalence and analysis of potential risk factors for Cryptosporidium parvum infection in lambs in Zaragoza (northeastern Spain).

An epidemiologic study was carried out to investigate the prevalence of and to identify factors associated with the risk of Cryptosporidium infection in sheep in Zaragoza (northeastern Spain). Faecal samples from 583 lambs aged from 1 day to 3 months and 205 ewes older than 1 year were collected at 89 farms in the two regions of the province of Zaragoza with the highest sheep population (Zaragoza and Ejea de los Caballeros). In every sheep farm, data of the factors potentially associated with the likelihood of C. parvum infection were analysed: geographical location, season, size of herd, number of lambs in the herd at sampling time, lambing period, cleaning of lambing area and presence of diarrhoeic lambs in the farm. C. parvum oocysts were identified by using the Ziehl-Neelsen technique in 344 lambs (59%) from 75 farms (84.4%). Infected lambs ranged from less than 7 days to 90 days of age, although the percentage of animals shedding oocysts peaked at 8-14 days of age (76.2%). Statistical analysis showed that infection rates were significantly higher in lambs aged between 1 and 21 days (66.4%) than in those aged between 22 and 90 days (23%) (P<0.0001, chi(2)). Analysis of correlation between excretion of oocysts and diarrhoea revealed a relationship in all age groups and the probability of presenting diarrhoea was significantly higher for lambs shedding oocysts (86.3%) than for those which did not excrete the parasite (32.2%) (P<0.0001, chi(2)). Similarly, cryptosporidial infection rates were significantly higher in diarrhoeic (79.4%) than in non-diarrhoeic lambs (22.4%). Furthermore, infection intensity was correlated with the presence of clinical symptoms. Presence of diarrhoeic lambs in the farm was the only factor significantly associated with an increased risk of infection since the percentage of herds testing positive was significantly higher in farms with diarrhoeic lambs (91.3%) than in those without cases of neonatal diarrhoea (12.5%) (P<0.0001, chi(2)). Factors associated with a decreased risk of C. parvum infection in lambs included low numbers of lambs in the farm and cleaning of the lambing area. Additionally, lambs 8-14 days of age were less likely to be infected at the first lambing period and in spring/autumn. Cryptosporidial infection was also detected in 16 ewes (7.8%) which excreted few oocysts and without diarrhoea.

Serum antibody response and Cryptosporidium parvum oocyst antigens recognized by sera from naturally infected sheep.

The response of specific serum immunoglobulins (IgG, IgM and IgA) and the major antigens of Cryptosporidium parvum recognized by these isotypes were investigated by using enzyme-linked immunosorbent assay and immunoblot techniques in lambs and ewes naturally infected throughout an outbreak of cryptosporidiosis. Serum samples were collected from 20 lambs the first day they showed diarrhoea (D1), and Days 11 and 22, in addition to single serum samples from 17 of their dams. Serum anti-C. parvum IgG, IgM and/or IgA antibodies were detected in lambs as early as Day 1. Levels of IgM antibodies remained steady from D1 to D11 and increased at D22, whereas the IgG response decreased from D1 to D11 and subsequently increased. In contrast, IgA antibodies rapidly fell from D1 and all lambs were seronegative at D11 and D22. The highest levels of specific antibodies were detected in sera from ewes. In fact, all ewes were seropositives for IgM and IgA isotypes and most (16/17) showed positive levels of IgG. Four protein fractions (37-39, 42-48, 51-57 and 60-69 kDa) were the most frequently recognized by IgG and IgM from lamb sera. A low molecular weight fraction (12-14 kDa) reacting with IgG and IgA in most lamb sera was scarcely recognized by IgM and three broad bands were frequently recognized by IgA antibodies (23-25, 51-57 and 90-95 kDa). The recognition pattern of 23-25 kDa peptides by IgA from lamb sera clearly increased with the age. Peptides of 42-48, 51-57, 60-69 and 71-78 kDa were most frequently recognized by IgG and IgM from ewe sera. In relation to IgA antibodies from ewe sera, a frequent immunoreactivity was found with proteins in the intervals between 12 and 22 kDa as well as between 32 and 34 kDa and practically all sera reacted with fractions from 42 to 95 kDa.

Eimeria tenella: hsp70 expression during sporogony.

There is an increasing interest in identifying the parasite components involved in the maturation, development, and infectivity of intracellular protozoan parasites. In the present study, a heat shock protein (hsp) of the family of 70 kDa hsp (hsp70), which play important roles in the stage conversion and virulence of these parasites, was examined. Whereas hsp70 expression has been examined in Eimeria tenella within host tissues, in the present study, oocysts of E. tenella were used to investigate the expression of hsp70 during sporulation without interference from the host; hsp70 expression during excystation was induced by incubating sporulated oocysts under various experimental conditions to produce the stimuli necessary for sporozoites to become active and to excyst in vitro. Hsp70 was detected by immunohistochemical techniques; quantitative flow cytometric analysis was also been carried out using specific monoclonal antibodies against hsp70. Hsp70 was expressed during sporulation but was not found in sporulated oocysts after the completion of sporulation. Oocysts re-expressed hsp70 when excystation was induced. The presence of hsp70 prior to infection may preadapt the parasite for additional stress in the host and may be involved in the formation of sporozoites.

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.