Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis

The sensitivity of LAMP, PCR and in vitro culture methods for the detection of Theileria equi and Babesia caballi was evaluated using tenfold serially diluted culture parasites. On day 1 post-culture, both T. equi and B. caballi parasites could only be observed at 1% parasite dilution from the in vitro culture method, whereas LAMP could detect up to 1 × 10⁻³% of both T. equi and B. caballi parasite dilutions, whilst PCR could detect 1 × 10⁻³% T. equi and 1 × 10⁻¹% B. caballi parasite dilutions. On day 7 post-culture, the detection limit for T. equi and B. caballi in the in vitro culture increased up to 1 × 10⁻⁶%, whereas LAMP detection limit increased to 1 × 10⁻¹⁰% for both parasites, whilst the PCR detection limit increased to 1 × 10⁻¹⁰% and 1 × 10⁻⁶% for T. equi and B. caballi, respectively. Furthermore, LAMP and PCR amplified the T. equi DNA extracted from the organs of an experimentally infected horse. This study further validates LAMP as an alternative molecular diagnostic tool, which can be used in the diagnosis of early infections of equine piroplasmosis and together with PCR can also be used as supplementary methods during post-mortems.     

An enzyme-linked immunosorbent assay for detection of Theileria parva antibodies in cattle using a recombinant polymorphic immunodominant molecule

Field and experimental bovine infection sera were used in immunoblots of sporozoite and schizont lysates of Theileria parva to identify candidate diagnostic antigens. Four parasite antigens of Mr 67,000 (p67), 85,000 (the polymorphic immunodominant molecule, PIM), 104,000 (p104), and 150,000 (p150) were selected for a more detailed analysis. The p67 and p104 antigens were present only in the sporozoite lysates, whereas PIM and p150 were found in both sporozoite and schizont lysates. The four antigens were expressed as recombinant fusion proteins and were compared with each other in an enzyme-linked immunosorbent assay (ELISA) and in the whole-schizont-based indirect fluorescent antibody test (IFAT) in terms of their ability to detect antibodies in sera of experimentally infected cattle. The PIM-based ELISA provided a higher degree of sensitivity and specificity than did the ELISA using the other three recombinant antigens or the IFAT. Further evaluation of the PIM-ELISA using experimental sera derived from cattle infected with different hemoparasites and field sera from endemic and nonendemic T. parva areas showed that the assay had a sensitivity of >99% and a specificity of between 94% and 98%. Received: 19 October 1997 / Accepted: 19 November 1997     

Efficacy of toltrazuril and ponazuril against experimental Neospora caninum infection in mice

Neosporosis is a disease affecting predominantly fetal development in cattle and dog hosts; and it may cause neuromuscular disfunction in infected new-born calves and pups. Predispositions – including, e.g. transient immunosuppression during pregnancy – may result in an increased dissemination of the parasite within the host or its offspring. Chemotherapeutic treatment of neosporosis may be an issue, provided that an appropriate drug is made available. In this respect, we describe the use of a mouse model for the evaluation of toltrazuril and ponazuril medication as a means of preventing parasite dissemination and subsequent formation of cerebral lesions. Toltrazuril- and ponazuril-treated mice were experimentally infected intraperitoneally (i.p.) with 2 × 10⁶ Neospora caninum tachyzoites. The infection was monitored at three levels: clinically, by assessing symptoms, histologically, by assessing the occurrence of cerebral lesions and parasites by immunohistochemistry, and on the molecular level, by detection of parasite DNA using PCR. Chemotherapy using either toltrazuril or ponazuril, both applied in a drinking-water formulation (20 mg toltrazuril or ponazuril kg⁻¹ body weight day⁻¹) completely prevented the formation of cerebral lesions in all treated animals, as assessed by immunohistochemistry. PCR analyses of these treated animals showed that DNA-detectability was reduced by 91% and 90% upon toltrazuril and ponazuril medication, respectively. Received: 6 August 2000 / Accepted: 7 August 2000     

Cross-protection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and RT-PCR and nested PCR for their detection

Summary.  A 1-step RT-PCR assay, targeting a 730 bp fragment of the nucleocapsid (N) gene of bovine coronavirus (BCV), and a nested PCR assay, targeting a 407 bp fragment of the N gene, were developed to detect BCV in nasal swab and fecal samples of calves experimentally exposed to BCV. Both 1-step RT-PCR and nested PCR recognized cell culture passaged isolates of 10 bovine respiratory coronavirus (BRCV), 5 calf diarrhea (CD) and 8 winter dysentery (WD) strains of BCV, but not transmissible gastroenteritis coronavirus or bovine rotavirus. The sensitivity of the 1-step RT-PCR and nested PCR was compared to that of an antigen-capture ELISA. The lowest detection limit of the 1-step RT-PCR and nested PCR as determined by using tenfold serial dilutions of the BRCV 255 and 440 strains in BCV negative nasal swab suspensions from preexposure gnotobiotic calves was 2 × 10⁴ and 2 × 10² TCID₅₀/0.1 ml for each strain, respectively. The lowest detection limit of the antigen-capture ELISA as determined by using the same serially diluted samples was 1 × 10⁶ TCID₅₀/0.1 ml for each strain. Therefore, the 1-step RT-PCR and nested PCR assays were 50 and 5000 times, respectively more sensitive than the antigen-capture ELISA to detect BRCV in nasal swab suspensions. To investigate in vivo cross-protection between the BRCV and CD or WD strains of BCV and to detect nasal and fecal shedding of BCV using the 1-step RT-PCR, nested PCR and antigen-capture ELISA, 6 colostrum-deprived and two gnotobiotic calves were inoculated with a BRCV, a CD or a WD strain of BCV and then challenged 3–4 weeks later with either BRCV, CD or WD strains of BCV. All calves developed diarrhea after inoculation and BCV antigen (ELISA) or RNA (RT-PCR) was detected in the diarrheic fecal samples or the corresponding nasal swab samples. In addition, low amounts of BCV were also detected only by nested PCR in the fecal and nasal swab samples before and after diarrhea. No respiratory clinical signs were observed during the entire experimental period, but elevated rectal temperatures were detected during diarrhea in the BCV-inoculated calves. All calves recovered from infection with the BRCV, CD, or WD strains of BCV were protected from BCV-associated diarrhea after challenge exposure with either a heterologous or homologous strain of BCV. However, all calves challenged with heterologous BCV strains showed subclinical BCV infection evident by detection of nasal and fecal shedding of BCV RNA detected only by nested PCR. Such results confirm field and experimental data documenting reinfection of the respiratory and enteric tracts of cattle, suggesting that, in closed herds, respiratory or enteric tract reinfections may constitute a source of BCV transmissible to cows (WD) or neonatal or feedlot calves. In addition, the present 1-step RT-PCR and nested PCR assays were highly sensitive to detect BCV in nasal swab and fecal specimens. Therefore, these assays should be useful to diagnose BCV infections in calves and adult cows. Received Septemper 21, 2000 Accepted January 17, 2001     

Detection and differentiation of ovine Theileria and Babesia by reverse line blotting in China

A reverse line blot (RLB) assay was developed for detection and specific identification of the different ovine Theileria and Babesia parasites. In a polymerase chain reaction (PCR), the hypervariable region 4 (V4 region) of the 18S ribosomal DNA gene was amplified with a set of general primers specific for members of the genera Theileria and Babesia. Meanwhile, specific oligonucleotide probes were designed and bound on membrane. After one single-PCR amplification, the amplified fragment was hybridized against different generic and species-specific probes. It was able to detect four species, i.e., Babesia motasi (Chengde, Lintan, Ningxian, Tianzhu), Babesia sp. (Kashi), Theileria luwenshuni (Lintan, Madang, Ningxian), Theileria uilenbergi (Longde, Zhangjiachuan) as defined previously. All probes bound to their respective target sequence only; therefore, no cross-reaction was observed, resulting in clear recognition of either individual strains, species, or groups in normal positive tests. Meanwhile, no signal was observed when ovine genomic DNA and water were used as a control, demonstrating that the signals are due to the presence of parasite DNA in the samples. Furthermore, the sensitivity of RLB could be considerably enhanced to detect a parasitemia level between10⁻³% and 10⁻⁸%. Finally, 117 samples from field were tested with RLB, PCR, and enzyme-linked immunosorbent assay (ELISA). The positive rate of RLB was higher than that of PCR and ELISA, and furthermore, RLB could determinate the species of piroplasms, the samples were infected with. Samples, 1,117, from five areas in Gannan Tibet Autonomous Region have been examined with RLB assay and compared with ELISA assay for corresponding samples. The results showed that the positive rate of RLB was higher than that of ELISA test obviously, and both T. luwenshuni and T. uilenbergi were widely distributed in these areas. RLB developed here could be used for differentiation of Babesia and Theileria infection and for epidemiological survey, which was difficult to achieve by classical methods. In conclusion, the RLB is a versatile technique for simultaneous detection and identification of all ovine piroplasms.     

Highly debilitating natural <Emphasis Type="Italic">Trypanosoma vivax</Emphasis> infections in Brazilian calves: epidemiology,pathology, and probable transplacental transmission

Clinical, epidemiological, and pathological aspects of trypanosomiasis caused by Trypanosoma vivax in calves were reported for the first time in northeast Brazil. Clinical and epidemiological data, packed cell volumes (PCV), and parasitemia were assessed in 150 calves in May 2009 (rainy season—survey 1) and in 153 calves in November 2009 (dry season—survey 2) in three farms (A, B, and C). Prevalence of T. vivax in calves examined in the survey 1 was 63.3%, 65.0%, and 80.0% in farms A, B, and C, respectively. Morbidity varied from 63.3% to 80%, mortality from 15% to 30% and lethality from 23% to 37.5%. In survey 1, for all farms, high parasitemia (from 30.3 to 26.2 × 10⁶ parasites/mL), fever (from 39.8 to 40.3°C), low PCV (from 15.7% to 18.1%), and body score (from 2.5 to 3.5) were detected. Calves showed depression, weight loss, pale mucous membranes, enlarged lymph nodes, edema of the dewlap, cough, coryza, and diarrhea. The animals from farms A and B were treated with diminazene aceturate. Six months after, in survey 2, non-treated calves from farm C showed values for prevalence (81.82), morbidity (81.82), mortality (12.73), and lethality (15.55) similar to those in survey 1 (P > 0.05). Also in survey 2, four calves aging merely 1–3 days old presented high parasitemia levels (from 32 × 10⁶ to 74 × 10⁶ parasites/mL), suggesting transplacental transmission. In conclusion, trypanosomiasis by T. vivax constitutes high prevalent disease for calves raised in Brazilian semiarid and may have transplacental transmission.     

A study on the prevalence of a tick-transmitted pathogen, Theileria annulata, and hematological profile of cattle from Southern Punjab (Pakistan)

The present study was carried out to determine the prevalence of Theileria annulata in large ruminants in Southern Punjab (Pakistan). Blood samples were collected from 144 large ruminants, consisting of 105 cattle and 39 buffaloes, from six districts of Southern Punjab including Multan, Layyah, Muzaffar Garh, Bhakar, Bahawalnagar, and Vehari. Data on the characteristics of the animals and herds were collected through questionnaires. The age of animals (P = 0.02), presence of ticks on animals (P = 0.02), and presence of ticks on dogs associated with herds (P = 0.05) were among the major risk factors involved in the spread of tropical theileriosis in the study area. Two different parasite detection techniques, PCR amplification and screening of Giemsa-stained slides, were compared, and it was found that PCR amplification is a more sensitive tool (19% parasite detection) as compared to smear scanning (3% parasite detection) for the detection of T. annulata. Twenty eight out of 144 animals produced the 721-bp fragment specific for T. annulata from five out of six sampling districts. Different blood (hemoglobin, glucose) and serum (ALT, AST, LDH, cholesterol) parameters of calves and cattle were measured and compared between parasite-positive and parasite-negative samples to assess the effect of T. annulata on the blood and serological profile of infected animals.     

Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model, the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10⁻² ng/μl (278-bp, S. enterica), 11.2 × 10⁻² ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10⁻² ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 10⁴ copies/μl, 3.58 × 10⁴ copies/μl, and 1.79 × 10⁴ copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA samples from different food sources in parallel, thereby achieving another level of multiplexing.     

Real-time PCR strategy and detection of bacterial agents of lymphadenitis

The aim of this study was to compare 16 S rRNA gene amplification and sequencing with a systematic real-time PCR assay screening strategy that includes all common known pathogens recovered from lymph node biopsy specimens. Lymph node biopsy samples sent to our laboratory from January 2007 to December 2008 were tested in the study. Lymph nodes were screened for the presence of any bacteria by PCR amplification and sequencing targeting the 16 S rRNA gene and also by a specific real-time PCR strategy that includes Bartonella henselae, mycobacteria, Francisella tularensis, and Tropheryma whipplei. By testing 491 lymph nodes, we found that the sensitivity of our specific real-time PCR assay strategy was significantly higher than 16 S rRNA PCR amplification and sequencing for the detection of Bartonella henselae (142 vs 98; p < 10⁻⁴), Francisella tularensis (16 vs 10, p < 10⁻⁴), and mycobacteria (8 versus 3, p < 10⁻⁴). None of the samples was positive for Tropheryma whipplei. Our study demonstrates the usefulness and specificity of a systematic real-time PCR strategy for molecular analysis of lymph node biopsy specimens and the higher sensitivity compared with standard 16 S rRNA gene amplification and sequencing.     

Anti-<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Antibodies are Frequent in Type 1 Diabetes

Anti-Saccharomyces cerevisiae antibodies (ASCA) have been described in many autoimmune diseases in which there is an increased intestinal permeability. Also in type 1 diabetes (T1D), there is an increased intestinal permeability. Since no data are available about ASCA in T1D, we evaluated, retrospectively, the frequency of ASCA in this disease. ASCA, IgG, and IgA, were determined by ELISA in sera of 224 T1D patients in which coeliac disease has been excluded and 157 healthy control group. The frequency of ASCA (IgG or IgA) was significantly higher in T1D patients than in the control group (24.5% vs. 2.5%, p < 10⁻⁷). The same observation was found in children and in adult patients when we compare them to healthy children and blood donors group respectively. Compared to children, adult patients with T1D showed significantly higher frequencies of ASCA of any isotype (38% vs. 13.7%, p < 10⁻⁴), both ASCA IgG and IgA (12% vs. 1.6%, p = 0.002), ASCA IgG (35% vs. 9.8%, p < 10⁻⁵) and ASCA IgA (15% vs. 5.6%, p = 0.001). The frequency of ASCA was statistically higher in females of all T1D than in males (30.8% vs.17.7%, p = 0.03), in girls than in boys (22% vs.6.2%, p = 0.017), and significantly higher in men than in boys (35.7% vs. 6.2%, p < 10⁻⁴). The frequency of ASCA IgG was significantly higher than that of ASCA IgA in all T1D patients (21% vs. 9.8%, p < 0.002), in all females (26.5% vs. 10.2%, p < 0.002), in women (37.9% vs. 12%, p < 0.001). The frequency of ASCA was significantly higher in all long-term T1D than in an inaugural T1D (29% vs. 14.5%, p = 0.019). The same observation was found in adults (45.8% vs. 17.8%, p = 0.01). In long-term T1D patients, ASCA were significantly more frequent in adults than children (45.8% vs. 14.5%, p < 10⁻⁴). The frequency of ASCA IgG was significantly higher in long-term T1D than in an inaugural T1D (25.2% vs. 11.6%, p = 0.03). Patients with T1D had a high frequency of ASCA.     

Haematological and serum biochemical profile of the upside-down catfish, Synodontis membranacea Geoffroy Saint Hilaire from Jebba Lake, Nigeria

Haematological and serum biochemical studies of natural population of Synodontis membranacea from Jebba Lake, North Central Nigeria were investigated in order to establish their mean and reference values. Bi-monthly collection of 1,408 live fish samples was carried out between April 2002 and March 2004, using gill nets of various mesh sizes ranging from 5.08 to 10.16 cm. The mean baseline value established for species-specific haematological and serum biochemical parameters were red blood cell (RBC) 3.83 ± 1.49 × 10¹² l⁻¹, haemoglobin (HB) 8.38 ± 1.96 g dl⁻¹, and packed cell volume (PCV) 25.65 ± 5.89%; mean cell volume 78.25 ± 37.90 fl; mean cell haemoglobin (MCH) 33.04 ± 12.50 pg; mean cell haemoglobin concentration 26.53 ± 15.18 g dl⁻¹; white blood cell (WBC) 315.65 ± 95.37 × 10⁻⁹; agranulocytes (Agr) 82.07 ± 11.38%; monocytes (Mon) 6.37 ± 3.01%; lymphocytes (Lym) 76.49 ± 10.81%; granulocytes (Gran) 40.28 ± 17.48%; neutrophils (Neut) 24.42 ± 10.68%; eosinophils (Eos) 16.14 ± 8.25%; basophils 0.09 ± 0.04%; protein 40.19 ± 7.45 g l⁻¹; albumin 19.78 ± 5.67 g l⁻¹; creatinine 49.71 ± 16.15 μmol l⁻¹; urea 3.05 ± 0.67 nmol l⁻¹; uric acid 0.76 ± 0.33 nmol l⁻¹; glucose 4.24 ± 1.74 mmol l⁻¹; cholesterol 8.46 ± 2.27 mmol l⁻¹; calcium 2.35 ± 0.94 mmol l⁻¹; potassium 13.36 ± 4.45 mmol l⁻¹; sodium 139.39 ± 23.19 mmol l⁻¹; alanine aminotransferase (ALT) 11.79 ± 2.67 U l⁻¹; aspartate aminotransferase 16.80 ± 4.73 U l⁻¹; and alkaline phosphatase 63.01 ± 20.44 U l⁻¹. Only three of these parameters (i.e. neutrophil, glucose and potassium) differed significantly (P > 0.05) on gender basis. Pearson’s correlation coefficients indicated significant relationship of standard length and total weight with RBC, PCV, HB, WBC, Agr, Mon, Lym, Gran, Neut, Eos, sodium, and ALT only. The study has provided baseline haematological and biochemical data for use in health monitoring and productivity of S. membranacea, which would be of great value for future comparative surveys in this era of increased fish culture in Nigeria.     

Apoptosis Inhibitor Expressed by Macrophages Tempers Autoimmune Colitis and the Risk of Colitis-Based Carcinogenesis in TCRα<Superscript>−/−</Superscript> Mice

Background Crohn’s disease and ulcerative colitis (UC) are the two main entities involved in human inflammatory bowel disease (IBD). However, their precise etiologies remain unclear. To study the development of mucosal inflammation, and chronic inflammation-based dysplasia and carcinoma formation, we examined possible roles of the apoptosis inhibitor expressed by macrophages (AIM) in an experimental IBD model. Methods In this study, we used T cell receptor α deficient (TCRα^−/−) mice, a known UC-like colitis model. We generated TCRα^−/− × AIM^−/− double knockout mice by crossbreeding TCRα^−/− with AIM^−/− mice. At 24 weeks of age, mice were killed to obtain colon tissues for pathological examinations. TCRα^−/− × AIM^+/− mice, heterozygous littermates of TCRα^−/− × AIM^−/− mice, were used as controls. Results Severe colitis was observed in TCRα^−/− × AIM^−/− mice, when compared with TCRα^−/− × AIM^+/− mice. Dysplasia was detected in TCRα^−/− × AIM^−/− mice, but not in TCRα^−/− × AIM^+/− mice. Adenocarcinoma formation was observed from dysplasia only in TCRα^−/− × AIM^−/− mice. Conclusion Not only a high incidence of severe colitis but also dysplasia and adenocarcinoma formation were observed in TCRα^−/− × AIM^−/− mice only. AIM have some regulatory roles in inflammation and progression of dysplasia to carcinoma in TCRα^−/− mice.     

Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant ( Elephas maximus ) and Man ( Homo sapiens )

Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, P _d of water in RBC was measured by using a Mn²⁺-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of P _d were, in the case of elephant RBC, 3.2 × 10⁻³ cm/s at 25 °C, 3.9 × 10⁻³ cm/s at 30 °C, 5.2 × 10⁻³ cm/s at 37 °C and 6.5 × 10⁻³ cm/s at 42 °C; all values were significantly lower than the corresponding values of P _d for human RBC, namely 4.3 × 10⁻³ cm/s at 25 °C, 5.2 × 10⁻³ cm/s at 30 °C, 6.1 × 10⁻³ cm/s at 37 °C, 7.8 × 10⁻³ cm/s at 42°C. The maximal inhibition of P _d (56%) was reached in 30 min at 37 °C with 2 mm p-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10⁻³ cm/s for elephant and 2.6 × 10⁻³ cm/s for human RBC. The values of the activation energy for water permeability (E _a,d ) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.     

Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant (Elephas maximus) and Man (Homo sapiens)

Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, P _d of water in RBC was measured by using a Mn²⁺-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of P _d were, in the case of elephant RBC, 3.2 × 10⁻³ cm/s at 25 °C, 3.9 × 10⁻³ cm/s at 30 °C, 5.2 × 10⁻³ cm/s at 37 °C and 6.5 × 10⁻³ cm/s at 42 °C; all values were significantly lower than the corresponding values of P _d for human RBC, namely 4.3 × 10⁻³ cm/s at 25 °C, 5.2 × 10⁻³ cm/s at 30 °C, 6.1 × 10⁻³ cm/s at 37 °C, 7.8 × 10⁻³ cm/s at 42°C. The maximal inhibition of P _d (56%) was reached in 30 min at 37 °C with 2 mm p-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10⁻³ cm/s for elephant and 2.6 × 10⁻³ cm/s for human RBC. The values of the activation energy for water permeability (E _a,d ) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.     

Erythropoietin acute reaction and haematological adaptations to short, intermittent hypobaric hypoxia

This study aimed to determine whether brief hypoxic stimuli in a hypobaric chamber are able to elicit erythropoietin (EPO) secretion, and to effectively stimulate erythropoiesis in the short term. In two different experiments, a set of haematological, biochemical, haemorheological, aerobic performance, and medical tests were performed in two groups of healthy subjects. In the first experiment, the mean plasma concentration of EPO ([EPO]) increased from 8.7 to 13.5 mU · ml⁻¹ (55.2%; P < 0.01) after 90 min of acute exposure at 540 hPa, and continued to rise until a peak was attained 3 h after the termination of hypoxia. In the second experiment, in which subjects were exposed to a simulated altitude of up to 5500 m (504 hPa) for 90 min, three times a week for 3 weeks, all haematological indicators of red cell mass increased significantly, reaching the highest mean values at the end of the programme or during the subsequent 2 weeks, including packed cell volume (from 42.5 to 45.1%; P < 0.01), red blood cell count (from 4.55 × 10⁶ to 4.86 × 10⁶ · l⁻¹; P < 0.01), reticulocytes (from 0.5 to 1.4%; P < 0.01), and haemoglobin concentration (from 14.3 to 16.2 g · dl⁻¹; P < 0.01), without an increase in blood viscosity. Arterial blood oxygen saturation during hypoxia was improved (from 60% to 78%; P < 0.05). Our most relevant finding is the ability to effectively stimulate erythropoiesis through brief intermittent hypoxic stimuli (90 min), in a short period of time (3 weeks), leading to a lower arterial blood desaturation in hypoxia. The proposed mechanism for these haematological and functional adaptations is the repeated triggering effect of EPO production caused by the intermittent hypoxic stimuli. Accepted: 15 December 1999     

Assessment of Leishmania promastigote growth in vitro by means of nucleoside hydrolase activity determination

Nucleoside hydrolases (NH) are involved in the purine salvage pathway of protozoan cells for the biosynthesis of nucleic acids. We developed a simple and reliable microassay based on N-ribohydrolase dosage using 4-nitrophenyl-β-d-ribofuranoside (NPR) substrate for the quantification of Leishmania infantum. The free promastigote stage of L. infantum contains high amounts of NH capable of cleaving NPR, but the parasitic amastigote does not. The method allows reliable quantification of viable parasites over a wide range of concentrations (5 × 10⁴–2 × 10⁸ parasites ml⁻¹) in a single assay. No difference in NH activity was observed between various strains at equivalent concentrations and growth curves determined with NH dosage were similar to optical counts. Samples can be stored at −20 °C for weeks before use in this unique assay without significant loss of NH activity. The method is particularly simple and versatile and proves well adapted for the determination of growth characteristics and drug screening studies of L. infantum promastigotes in vitro. Received: 4 August 2000 / Accepted: 4 August 2000     

Physiological and metabolic responses of female games and endurance athletes to prolonged, intermittent, high-intensity running at 30° and 16°C ambient temperatures

Eight female games players (GP) and eight female endurance athletes (EA) ran intermittently at high-intensity and for prolonged periods in hot (30°C) and moderate (16°C) ambient temperatures. The subjects performed a two-part (A, B) test based on repeated 20-m shuttle runs. Part A comprised 60 m of walking, a maximal 15-m sprint, 60 m of cruising (90% maximal oxygen uptake, V˙O_2max) and 60 m of jogging (45% V˙O_2max) repeated for 75 min with a 3-min rest every 15 min. Part B involved an exercise and rest pattern of 60-s running at 100% V˙O_2max and 60-s rest which was continued until fatigue. Although the GP and EA did not respond differently in terms of distances completed, performance was 25 (SEM 4)% less (main effect trial, P < 0.01) in the hot (HT) compared with the moderate trial (MT). Sprints of 15 m took longer to complete in the heat (main effect, trial, P < 0.01), and sprint performance declined during HT but not MT (interaction, trial × time, P < 0.01). A very high correlation was found between the rate of rise in rectal temperature in HT and the distance completed [GP, r =−0.94, P < 0.01; EA (n = 7), r = −0.93, P < 0.01]. Blood lactate [La⁻ ]_b and plasma ammonia [NH₃]_p1 concentrations were higher for GP than EA, but were similar in HT and MT [La⁻ ]_b, HT: GP vs EA, 8.0 (SEM 0.9) vs 4.9 (SEM 1.1) mmol · l⁻¹; MT: GP vs EA, 8.0 (SEM 1.3) vs 4.4 (SEM 1.2) mmol · l⁻¹; interaction, group × time, P < 0.01; [NH₃]_p1, HT: GP vs EA, 70.1 (SEM 12.7) vs 43.2 (SEM 6.1) mmol · l⁻¹; MT: GP vs EA, 76.8 (SEM 8.8) vs 32.5 (SEM 3.8) μmol · l⁻¹; interaction, group × time, P < 0.01. Ad libitum water consumption was higher in HT [HT: GP vs EA, 18.9 (SEM 2.9) vs 13.5 (SEM 1.7) ml · kg⁻¹ · h⁻¹; MT: GP vs EA, 12.7 (SEM 3.7) vs 8.5 (SEM 1.5) ml · kg⁻¹ · h⁻¹; main effect, group, n.s.; main effect, trial, P < 0.01]. These results would suggest that elevated body temperature is probably the key factor limiting performance of prolonged, intermittent, high-intensity running when the ambient temperature is high, but not because of its effect on the metabolic responses to exercise. Accepted: 19 July 1999     

The effect of shear stress on the basolateral membrane potential of proximal convoluted tubule of the rat kidney

As consequence of glomerular filtration the viscosity of blood flowing through the efferent arteriole increases. Recently, we found that shear stress modulates proximal bicarbonate reabsorption and nitric oxide (NO·) was the chemical mediator of this effect. In the present work, we found that agonists of NO· production affected basolateral membrane potential (V _blm) of the proximal convoluted tubule (PCT) epithelium. Using paired micropuncture experiments, we perfused peritubular capillaries with solutions with different viscosity while registering the V _blm. Our results showed that a 50% increment in the viscosity, or the addition of bradykinin (10⁻⁵ M) to the peritubular perfusion solution, induced a significant and similar hyperpolarization of the V _blm at the PCT epithelium of 6 ± 0.7 mV (p < 0.05). Both hyperpolarizations were reverted by l-NAME (10⁻⁴ M). Addition of 2,2′-(hydroxynitrosohydrazino) bis-ethanamine (NOC-18) 3 × 10⁻⁴ M to the peritubular perfusion solution induced a hyperpolarization of the same magnitude of that high viscosity or bradykinin. These results strongly suggest the involvement of NO· in the effect of high viscosity solutions. This effect seems to be mediated by activation of channels as glybenclamide (5 × 10⁻⁵ M) added to peritubular solutions induced a larger depolarization of the V _blm with high viscosity solutions. Acetazolamide (5 × 10⁻⁵ M) added to high viscosity solutions induced a larger hyperpolarization (8 ± 1 mV; p < 0.05), suggesting that depolarizing current due to exit across the basolateral membrane damps the hyperpolarizing effect of high viscosity. Considering that Na⁺ and consequently water reabsorption is highly dependent on electrical gradient, the present data suggest that the endothelium of kidney vascular bed interacts in paracrine fashion with the epithelia, affecting V _blm and thus modulating PCT reabsorption.     

Effect of Lysophosphatidylcholine on α-Adrenoreactivity of Rat Aorta Smooth Muscles

In experiments on rat aortic ring segments, lysophosphatidylcholine in concentrations of 2 × 10⁻⁶, 2 × 10⁻⁵, and 2 × 10⁻⁴ M did not suppress the tonotropic effect of phenylephrine (6 × 10⁻⁶ and 6 × 10⁻⁵ M) and in concentration of 2 × 10⁻⁵ M even potentiated it, which was noted for phenylephrine at a concentration of 6 × 10⁻⁶ M. It was concluded that the chemomodulating effect of lysophosphatidylcholine depends on the type of receptors and cells.     

Measurement and prediction of peak shivering intensity in humans

Prediction equations of shivering metabolism are critical to the development of models of thermoregulation during cold exposure. Although the intensity of maximal shivering has not yet been predicted, a peak shivering metabolic rate (Shiv_peak) of five times the resting metabolic rate has been reported. A group of 15 subjects (including 4 women) [mean age 24.7 (SD 6) years, mean body mass 72.1 (SD 12) kg, mean height 1.76 (SD 0.1) m, mean body fat 22.3 (SD 7)% and mean maximal oxygen uptake (V˙O_2max) 53.2 (SD 9) ml O₂ · kg⁻¹ · min⁻¹] participated in the present study to measure and predict Shiv_peak. The subjects were initially immersed in water at 8°C for up to 70 min. Water temperature was then gradually increased at 0.8 °C · min⁻¹ to a value of 20 °C, which it was expected would increase shivering heat production based on the knowledge that peripheral cold receptors fire maximally at approximately this temperature. This, in combination with the relatively low core temperature at the time this water temperature was reached, was hypothesized would stimulate Shiv_peak. Prior to warming the water from 8 to 20 °C, the oxygen consumption was 15.1 (SD 5.5) ml · kg⁻¹ · min⁻¹ at core temperatures of approximately 35 °C. After the water temperature had risen to 20 °C, the observed Shiv_peak was 22.1 (SD 4.2) ml O₂ · kg⁻¹ · min⁻¹ at core and mean skin temperatures of 35.2 (SD 0.9) and 22.1 (SD 2.2) °C, respectively. The Shiv_peak corresponded to 4.9 (SD 0.8) times the resting metabolism and 41.7 (SD 5.1)% of V˙O_2max. The best fit equation predicting Shiv_peak was Shiv_peak (ml O₂ · kg⁻¹ · min⁻¹)=30.5 + 0.348 ×V˙O_2max (ml O₂ · kg⁻¹ · min⁻¹) − 0.909 × body mass index (kg · m⁻²) − 0.233 × age (years); (P=0.0001; r ²=0.872). Accepted: 7 September 2000