The innate resistance of Trypanosoma copemani to human serum.

Trypanosoma copemani is known to be infective to a variety of Australian marsupials. Characterisation of this parasite revealed the presence of stercorarian-like life-cycle stages in culture, which are similar to T. rangeli and T. cruzi. The blood incubation infectivity test (BIIT) was adapted and used to determine if T. copemani, like T. cruzi and T. rangeli, has the potential to grow in the presence of human serum. To eliminate any effects of anticoagulants on the complement system and on human high density lipoprotein (HDL), only fresh whole human blood was used. Trypanosoma copemani was observed by microscopy in all human blood cultures from day 5 to day 19 post inoculation (PI). The mechanism for normal human serum (NHS) resistance in T. copemani is not known. The results of this study show that at least one native Australian trypanosome species may have the potential to be infective for humans.

Evaluation of the implementation of the Xpert® MTB/RIF assay in Fiji.

SETTING: All Xpert® MTB/RIF tests performed in the three TB (tuberculosis) treatment centres in Fiji from June 2012 to February 2013. OBJECTIVES: To determine 1) the number of Xpert tests performed in each centre, 2) the association between sputum quality and Xpert results, 3) the agreement of Xpert with acid-fast bacilli (AFB) smear microscopy and TB culture and 4) error rates. DESIGN: Retrospective review of records. RESULTS: A total of 415 Xpert tests were performed in the study period. Mycobacterium tuberculosis was detected in 69 (16.6%) samples. No rifampicin resistance was detected. M. tuberculosis was detected from 60 (18.7%) good-quality sputum samples. A total of 43 (10.4%) errors occurred during this period. M. tuberculosis was detected in 10 (2.9%) smear-negative specimens. There was a substantial and an almost perfect agreement between Xpert and AFB microscopy (κ = 0.793) and culture results (κ = 0.818), respectively. CONCLUSION: Although a good correlation between Xpert and the two tests were shown in the study, Xpert should not replace the routine first-line TB diagnostic tests used in Fiji for reasons related to logistics and sustainability. A further evaluation of the assay's performance is required over a longer time period to gauge its diagnostic value in detecting smear-negative, Xpert-positive cases in Fiji.

Contexte : Tous les tests Xpert® MTB/RIF réalisés dans les trois centres de traitement anti-tuberculeux aux Fidji entre juin 2012 et février 2013.Objectifs : Déterminer 1) le nombre de tests Xpert réalisés dans chaque centre, 2) l'association entre la qualité des crachats et le résultat du test Xpert, 3) l'accord entre Xpert et la microscopie des crachats acido alcoolo résistant (AFB) et la culture, et 4) le taux d'erreurs.Schéma : Revue rétrosp ective de dossiers.Résultats : Un total de 415 tests Xpert a été réalisé pendant la période d'étude. Mycobacterium tuberculosis a été détecté dans 69 (16.6%) échantillons. Aucune résistance à la rifampicine n'a été décelée. M. tuberculosis a été identifié dans 60 (18,7%) échantillons de crachats de bonne qualité. Un total de 43 (10,4%) erreurs sont survenues pendant la période d'étude. M. tuberculosis a été identifié dans 10 (2,9%) spécimens à frottis négatif. Il y a eu une concordance substantielle et presque parfaite entre les résultats du Xpert et ceux de la microscopie AFB (κ = 0,793) et de la culture (κ = 0,818), respectivement.Conclusion : En dépit de la bonne corrélation entre Xpert et les deux autres tests mise en évidence dans l'étude, Xpert ne peut toujours pas remplacer les tests de diagnostic de routine utilisés en première intention aux Fidji, en raison de contraintes logistiques et de problèmes de pérennité. Il est nécessaire de réaliser une évaluation ultérieure de la performance de ce test sur une période plus longue afin de mesurer sa valeur diagnostique dans la détection de cas à frottis négatif, Xpert positif aux Fidji.

Marco de referencia: Todas las pruebas Xpert® MTB/RIF realizadas en los tres centros de atención de la tuberculosis (TB) de Fiji entre junio del 2012 y febrero del 2013.Objetivos: Obtener la siguiente información: 1) el número de pruebas Xpert realizadas en cada centro; 2) la asociación entre la calidad de la muestra de esputo y el resultado de la prueba Xpert; 3) la concordancia de los resultados de esta prueba con la baciloscopia y el cultivo para Mycobacterium tuberculosis; y 4) las tasas de error de la prueba.Método: Fue este un estudio retrospectivo a partir de las historias clínicas.Resultados: Durante el período del estudio se practicaron 415 pruebas Xpert. Esta prueba aportó un resultado positivo para M. tuberculosis en 69 muestras de esputo (16,6%). No se encontró resistencia a rifampicina. Se detectó M. tuberculosis en 60 de muestras de esputo de buena calidad (18,7%). Se registraron 43 resultados de error de la prueba durante el período estudiado (10,4%). La prueba detectó M. tuberculosis en 10 muestras con baciloscopia negativa (2,9%). La concordancia de los resultados de la prueba Xpert con la baciloscopia del esputo fue notable (κ = 0,793) y casi perfecta con los resultados del cultivo (κ = 0,818).Conclusión: Si bien en el presente estudio la prueba Xpert exhibió una buena correlación con los métodos de referencia, todavía no puede remplazar las pruebas sistemáticas de elección en el diagnóstico de la TB en Fiji por razones operativas y de sostenibilidad. Es necesario realizar nuevos estudios de rendimiento diagnóstico durante un período más prolongado, a fin de estimar su utilidad diagnóstica en los casos que presentan una baciloscopia negativa y una prueba Xpert positiva en Fiji.

Tuberculosis diagnostics in Fiji: how reliable is culture?

SETTINGS: Acid-fast bacilli (AFB) smear microscopy and Mycobacterium tuberculosis culture are the first-line diagnostic tests for tuberculosis (TB). The contamination of TB cultures significantly reduces the reliability of TB diagnosis. OBJECTIVE: To investigate factors associated with TB culture contamination in Fiji, and the relative diagnostic performance of culture compared to microscopy. DESIGN: All tests performed at the Daulakao Mycobacterium Reference Laboratory (DMRL) in Fiji from 2010 to 2012 were reviewed. Study variables included AFB smear and TB culture results, age and type of specimen, referring TB testing centre and patient age. RESULTS: Of 5708 specimens reviewed, 70% had both AFB smear and culture results recorded; 421 specimens were contaminated; 2.7% of specimens were either degraded or had no result recorded. There was moderate agreement (κ = 0.577) between the two tests. Culture was more likely to be positive at higher AFB smear scores. Culture contamination was associated with distance from the DMRL, sample age and operator-associated factors. CONCLUSION: Increases in the speed of referral from TB testing centres or the addition of preservatives to sputum specimens may results in less culture contamination. The planned introduction of liquid culture techniques in combination with culture on Ogawa media is likely to increase the sensitivity of TB diagnosis in Fiji.

Contexte : L'examen microscopique de crachats à la recherche de bacilles acido-alcoolo résistants (AFB) et la culture de Mycobacterium tuberculosis sont des examens de première intention dans le diagnostic de la tuberculose (TB). La contamination des cultures de TB réduit la fiabilité du diagnostic.Objectif: Examiner les facteurs associés à la contamination des cultures de TB aux Fidji et les performances relatives de la culture et de l'AFB.Méthodes : Tous les examens réalisés au Laboratoire de Référence des Mycobacterium de Daulakao aux Fidji de 2010 à 2012 ont été inclus. Les variables comprenaient les résultats de l'AFB et de la culture, l'âge et le type de spécimen, le centre d'examen ayant envoyé l'échantillon et l'âge du patient.Résultats: Des 5708 spécimens enregistrés, 70% comprenaient à la fois les résultats du frottis AFB et de la culture. Une contamination est survenue dans 421 spécimens et 2,7% d'entre eux étaient soit dégradés, soit sans résultats. L'accord entre les deux tests était modéré (κ = 0,577). La culture avait plus de chances d'être positive quand le score du frottis AFB était plus élevé. La contamination de la culture était associée à la distance par rapport au laboratoire de référence, à l'âge de l'échantillon et à des facteurs liés à l'opérateur.Conclusion : Une plus grande rapidité de transmission des échantillons de crachats depuis le centre d'examens anti-tuberculeux ou l'adjonction de conservateurs aux spécimens de crachats pourrait amener une diminution du taux de contamination des cultures. L'introduction de techniques de cultures liquides combinée à la culture sur milieu Ogawa augmentera la sensibilité du diagnostic de la TB aux Fidji.

Marco de Referencia: El examen microscópico en busca de bacilos acidorresistentes (AFB) y el cultivo de Mycobacterium tuberculosis constituyen las pruebas de elección en el diagnóstico de la tuberculosis (TB). Sin embargo, la contaminación de los cultivos disminuye la fiabilidad del diagnóstico.Objetivo: Investigar los factores que se asocian con la contaminación de los cultivos de M. tuberculosis en Fiji y el desempeño relativo del cultivo y la AFB.Metodos: Se incluyeron en el estudio todas las pruebas realizadas en el Daulako Mycobacterium Reference Laboratory (DMRL) en Fiji del 2010 al 2012. Las variables examinadas fueron el resultado de la AFB y del cultivo, el tipo de muestra, el lapso transcurrido desde la recogida de la muestra hasta la prueba, el centro de TB que la remitió y la edad del paciente.Resultados: Se registraron 5708 muestras; el 70% de los casos comportaba el resultado de la AFB y el cultivo. Se contaminaron 421 muestras y 2,7% de las muestras se degradaron o carecían de resultado. Se observó una concordancia moderada entre ambas pruebas (κ = 0,577). El cultivo fue con mayor frecuencia positivo en las muestras que presentaban una alta puntuación de AFB. La contaminación del cultivo se asoció con la distancia entre el centro de origen y el DMRL, el lapso transcurrido desde la recogida de la muestra y otros factores propios del operador.Conclusión: Al aumentar la velocidad de remisión de las muestras desde el centro de pruebas de TB o agregar conservantes a las muestras de esputo se puede disminuir la tasa de contaminación de los cultivos. La introducción de las técnicas de cultivo en medio líquido, además de la utilización del medio de Ogawa, permitirá mejorar la sensibilidad del diagnóstico de la TB en Fiji.

Product state and speed distributions in photochemical triple fragmentations.

The clearest dynamical signature of a roaming reaction is a very cold distribution of energy into the rotational and translational degrees of freedom of the roaming donor fragment (e.g. CO) and an exceptionally hot vibrational distribution in the roaming acceptor fragment (e.g. H2, CH4). These signatures were initially identified in joint experimental/theoretical investigations of roaming in H2CO and CH3CHO and are now used to infer the presence of roaming mechanisms in other photodissociation reactions. In this paper we construct a phase space theory (PST) model of triple fragmentation (3F) and show that the dynamical signature of 3F is similar to that of the roaming donor fragment. The PST model starts with a calculation of two-body fragmentation (2F) of a generic molecule, ABC into AB + C. Every AB fragment with sufficient energy to undergo subsequence spontaneous dissociation is allowed to dissociate and the PST distribution of energy into A + B products is calculated for every initial AB state. Using CH3CHO --> HCO + CH3 --> H + CO + CH3 as an example, we calculate that the energy disposal into the rotational and translational degrees of freedom of the 3F products is very low, and is similar to the dynamical signature expected for production of CO via a roaming mechanism. We compare the 3F PST model with published experimental data for photodissociation of CH3CHO and CH3OCHO at energies above the 3F threshold.

Evaluation of serological tests for H5N1 avian influenza on field samples from domestic poultry populations in Vietnam: consequences for surveillance.

In Vietnam, serological post H5N1 vaccination surveillance using the HI test is applied to assess the efficiency of the vaccination in addition to virological monitoring. In this paper we report on the evaluations of the performances of the haemagglutination inhibition (HI) test and of a H5-ELISA, using chicken and duck field samples. The evaluations were conducted by comparison with a pseudotyped-based virus neutralization test (H5pp VNT) performed in a reference laboratory and considered as a "gold standard" and also by using methods developed for imperfect reference test. Their global accuracy and best cut-offs were also estimated. Results from the HI test for several haemagglutinin subtypes and from a commercial type A influenza competition ELISA were also compared. The results showed that performance of the HI test was very good in comparison with the H5pp VNT. Data also clearly supported the cut-off of ≥ 4 log(2) used for the HI test for chickens but, a 3 log(2) positivity cut-off would be more appropriate for ducks. When compared with the VNT, the H5-ELISA showed poor specificity when using the positivity cut-off specified by the manufacturer but could be used as a screening test if confirmed by the HI test or the H5ppVNT which presents some interests for large scale testing (no need for biosafety level 3 conditions and high performance). A general and highly sensitive pre-screening can also be achieved using the detection of NP-specific antibodies with a competition ELISA. This appears of little interest in a context of high subtypes diversity where only a subtype is targeted for surveillance and control.

Microsatellite typing and population structuring of Trypanosoma evansi in Mindanao, Philippines.

Trypanosoma evansi, a blood-borne protozoan parasite with an extensive geographical range is the causative agent of the livestock disease known as surra. A total of 140 out of 179 T. evansi isolates collected between 2006 and 2007 from 44 villages (comprising of 16 reported surra outbreaks) in 3 provinces (Agusan del Sur (ADS), Surigao del Sur (SDS) and Agusan del Norte (ADN)) in Mindanao, Philippines were each successfully genotyped using a suite of 7 polymorphic microsatellites. The study identified 16 multi locus genotypes (MLG) within the T. evansi isolates and evidence of the spread of surra outbreaks from one village to another, most likely due to the movement of infected animals. Genotyping provided evidence of population sub-structuring with 3 populations (I, II and III (only 1 isolate)) identified. The most abundant population was II, which was the predominant population in ADS and SDS (p=0.022). In addition, buffalo mortality was statistically higher in outbreak areas associated with isolates from population I (13.6%) than with isolates from population II (6.9%) (p=0.047). The present study has highlighted the utility of microsatellite loci to improve understanding of the epidemiology of T. evansi and in tracking surra outbreaks.

Preserving new anthelmintics: a simple method for estimating faecal egg count reduction test (FECRT) confidence limits when efficacy and/or nematode aggregation is high.

As it has been 30 years since a new anthelmintic class was released, it is appropriate to review management practices aimed at slowing the development of anthelmintic resistance to all drug classes. Recommendations to delay anthelmintic resistance, provide refugia and the use of a simulation model were reviewed to find optimum treatment strategies that maintain nematode control. Simulated Australian conditions indicated that a common successful low-risk treatment program was a rapid rotation between a "triple-combination" product (benzimidazole+levamisole+abamectin) and a new high-efficacy drug (monepantel). Where Haemonchus contortus was a threat, moxidectin was required at critical times because of its persistent activity against this parasite. Leaving up to 4% of adult sheep untreated provided sufficient "refugia" for non-selected worms to reduce the risk of selecting for anthelmintic resistance without compromising nematode control. For a new anthelmintic, efficacy estimated by faecal egg count reduction (FECR) is likely to be at or close to 100%, however using current methods the 95% confidence limits (CL) for 100% are incorrectly determined as 100%. The fewer eggs counted pre-treatment, the more likely an estimate of 100% will occur, particularly if the true efficacy is >90%. A novel way to determine the lower-CL (LCL) for 100% efficacy is to reframe FECR as a binomial proportion, i.e. define: n and x as the total number of eggs counted (rather than eggs per gram of faeces) for all pre-treatment and post-treatment animals, respectively; p the proportion of resistant eggs is p = x/n and percent efficacy is 100 ×(1-p) (assuming equal treatment group sizes and detection levels, pre- and post-treatment). The LCL is approximated from the cumulative inverse beta distribution by: 95%LCL=100 ×(1-(BETAINV(0.975, x+1, n-x+1))). This method is simpler than the current method, independent of the number of animals tested, and demonstrates that for 100% efficacy at least 37 eggs (not eggs per gram) need to be counted pre-treatment before the LCL can exceed 90%. When nematode aggregation is high, this method can be usefully applied to efficacy estimates lower than 100%, and in this case the 95% upper-CL (UCL) can be estimated by: 95% UCL = 100 ×(1((BETAINV(0.025, x+1, n-x+1))), with the LCL approximated as described above. A simulation study to estimate the precision and accuracy of this method found that the more conservative 99%CL was optimum; in this case 0.975 and 0.025 are replaced by 0.995 and 0.005 to estimate the LCL and UCL, respectively.

Vector of Trypanosoma copemani identified as Ixodes sp.

A total of 41 ticks were collected from 15 quokkas on Bald Island and 2 ticks from a Gilbert's potoroo from Two Peoples Bay. Three species of Ixodid ticks Ixodes australiensis, Ixodes hirsti and Ixodes myrmecobii were identified on the quokkas known to have a high prevalence of Trypanosoma copemani. Tick faeces from ticks isolated from 8 individual quokkas and a Gilbert's potoroo were examined with one identified as positive for trypanosomes. Faecal examination revealed trypanosomes similar to in vitro life-cycle stages of T. copemani. In total 12 ticks were dissected and trypanosomes found in sections of their midgut and haemolymph, 49 and 117 days after collection. Tick faeces, salivary glands and midguts from I. australiensis were screened using an 18S rRNA PCR with amplification seen only from the midguts. Sequencing showed 100% homology to T. copemani (genotype A) and 99.9% homology to the wombat (AII) isolate of T. copemani. Trypanosomes were only detected in I. australiensis as neither I. hirsti nor I. myrmecobii survived the initial 30-day storage conditions. We therefore identify a vector for T. copemani as I. australiensis and, given the detection of trypanosomes in the faeces, suggest that transmission is via the faecal-oral route.

The potential impact of native Australian trypanosome infections on the health of koalas (Phascolarctos cinereus).

Whole blood collected from koalas admitted to the Australian Zoo Wildlife Hospital (AZWH), Beerwah, QLd, Australia, during late 2006-2009 was tested using trypanosome species-specific 18S rDNA PCRs designed to amplify DNA from Trypanosoma irwini, T. gilletti and T. copemani. Clinical records for each koala sampled were reviewed and age, sex, blood packed cell volume (PCV), body condition, signs of illness, blood loss, trauma, chlamydiosis, bone marrow disease, koala AIDS and hospital admission outcome ('survival'/ 'non-survival') were correlated with PCR results. Overall 73.8% (439/595) of the koalas were infected with at least 1 species of trypanosome. Trypanosoma irwini was detected in 423/595 (71.1%), T. gilletti in 128/595 (21.5%) and T. copemani in 26/595 (4.4%) of koalas. Mixed infections were detected in 125/595 (21%) with co-infections of T. irwini and T. gilletti (101/595, 17%) being most common. There was a statistical association between infection with T. gilletti with lower PCV values and body condition scores in koalas with signs of chlamydiosis, bone marrow disease or koala AIDS. No association between T. gilletti infection and any indicator of health was observed in koalas without signs of concurrent disease. This raises the possibility that T. gilletti may be potentiating other disease syndromes affecting koalas.

Novel trypanosome Trypanosoma gilletti sp. (Euglenozoa: Trypanosomatidae) and the extension of the host range of Trypanosoma copemani to include the koala ( Phascolarctos cinereus).

Trypanosoma irwini was previously described from koalas and we now report the finding of a second novel species, T. gilletti, as well as the extension of the host range of Trypanosoma copemani to include koalas. Phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated that T. gilletti was genetically distinct with a genetic distance (± s.e.) at the 18S rDNA locus of 2.7 ± 0.5% from T. copemani (wombat). At the gGAPDH locus, the genetic distance (± s.e.) of T. gilletti was 8.7 ± 1.1% from T. copemani (wombat). Trypanosoma gilletti was detected using a nested trypanosome 18S rDNA PCR in 3/139 (∼2%) blood samples and in 2/29 (∼7%) spleen tissue samples from koalas whilst T. irwini was detected in 72/139 (∼52%) blood samples and T. copemani in 4/139 (∼3%) blood samples from koalas. In addition, naturally occurring mixed infections were noted in 2/139 (∼1.5%) of the koalas tested.

Spectroscopic and computational studies of the laser photolysis of matrix isolated 1,2-dibromoethanes: formation and fate of the bromoethyl radicals.

We report experimental and computational studies of the photolysis of atmospherically important 1,2-dibromoethanes (1,2-C(2)X(4)Br(2); X = H, F) in Ar matrixes at 5 K. Using the pulsed deposition method, we find that significant conformational relaxation occurs for 1,2-C(2)H(4)Br(2) (EDB; observed anti/gauche ratio =30:1) but not for 1,2-C(2)F(4)Br(2) (TFEDB; anti/gauche = 3:1), which is traced to a larger barrier to rotation about the C-C bond in the latter. Laser photolysis of matrix-isolated EDB at 220 nm reveals the growth of infrared bands assigned to the gauche conformer and C(2)H(4)-Br(2) charge transfer complex (both as major products), and the C(2)H(4)Br radical and C(2)H(3)Br-HBr complex as minor (trace) products. The presence of the C(2)H(4)-Br(2) complex is confirmed in the UV/visible spectrum, which shows an intense charge transfer band at 237 nm that grows in intensity upon annealing. In contrast to previous reports, our experimental and computational results do not support a bridged structure for the C(2)H(4)Br radical in either the gas phase or matrix environments. We also report on the laser photolysis of matrix-isolated TFEDB at 220 nm. Here, the dominant photoproducts are the anti and gauche conformers of the C(2)F(4)Br radical, the vibrational and electronic spectra of which are characterized here for the first time. The increase in yield of radical for TFEDB vs EDB is consistent with the stronger C-Br bond in the fluoro-substituted radical species. The photochemistry of the C(2)F(4)Br radical following excitation at 266 nm was investigated and found to lead C-Br bond cleavage and formation of C(2)F(4). The implications of this work for the atmospheric and condensed phase photochemistry of the alkyl halides is emphasized.

Trypanosoma irwini n. sp (Sarcomastigophora: Trypanosomatidae) from the koala ( Phascolarctos cinereus).

The morphology and genetic characterization of a new species of trypanosome infecting koalas (Phascolarctos cinereus) are described. Morphological analysis of bloodstream forms and phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated this trypanosome species to be genetically distinct and most similar to Trypanosoma bennetti, an avian trypanosome with a genetic distance of 0.9% at the 18S rDNA and 10.7% at the gGAPDH locus. The trypanosome was detected by 18S rDNA PCR in the blood samples of 26 out of 68 (38.2%) koalas studied. The aetiological role of trypanosomes in koala disease is currently poorly defined, although infection with these parasites has been associated with severe clinical signs in a number of koalas. Based on biological and genetic characterization data, this trypanosome species infecting koalas is proposed to be a new species Trypanosome irwini n. sp.

Morphological and molecular characterization of Trypanosoma copemani n. sp. (Trypanosomatidae) isolated from Gilbert's potoroo ( Potorous gilbertii) and quokka ( Setonix brachyurus).

Little is known of the prevalence and life-cycle of trypanosomes in mammals native to Australia. Native Australian trypanosomes have previously been identified in marsupials in the eastern states of Australia, with one recent report in brush-tailed bettongs (Bettongia penicillata), or woylie in Western Australia in 2008. This study reports a novel Trypanosoma sp. identified in blood smears, from 7 critically endangered Gilbert's potoroos (Potorous gilbertii) and 3 quokkas (Setonix brachyurus) in Western Australia. Trypanosomes were successfully cultured in vitro and showed morphological characteristics similar to members of the subgenus Herpetosoma. Phylogenetic analysis of 18S rRNA gene sequences identified 2 different novel genotypes A and B that are closely related to trypanosomes previously isolated from a common wombat (Vombatus ursinus) in Victoria, Australia. The new species is proposed to be named Trypanosoma copemani n. sp.

Estimating the impact of Trypanosoma evansi infection (surra) on buffalo population dynamics in southern Philippines using data from cross-sectional surveys.

Despite the widespread problem with surra (Trypanosoma evansi) in livestock, there are no published studies on its impact on host populations, probably because of the large financial and time cost involved in performing longitudinal studies. During 2002-6, a cross-sectional survey for T. evansi infection involving 1732 buffaloes from 71 villages in southern Philippines was carried out. Other livestock animals (horses, cattle and goats) in every surveyed village were also tested for infection with T. evansi but domestic buffaloes were the primary survey target. Seroprevalence ranged from 6% to 21% and 13% to 100% for buffaloes in low and high risk areas, respectively. Key demographic parameters were estimated from the age structured distributions of the sampled buffalo population for each sex. All areas were dominated by females (69%) and the annual calving rate for areas of 100% and low seroprevalence was 15% and 47%, respectively. Males were removed at a relatively high annual rate of 27% in all areas. In the main reproductive years (4-10) female removal/mortality was <1% and 10% for low and high risk areas, respectively. Older females were removed/died at a rate similar to males regardless of area. In high risk areas there were consistently more 2-year than 1-year old females and the reverse was true for the low risk areas. This implies that females were imported to the high risk areas for breeding. By assuming a stable age structure and similar size populations in each area, it was estimated that 28% of female calves need to be moved from low to high risk areas to maintain the observed age structure. In high risk areas, surra imposes significant financial losses due to reduced fertility, high mortality/removal rate and the necessity to import replacement buffaloes.

Models for Trypanosoma evansi (surra), its control and economic impact on small-hold livestock owners in the Philippines.

Simple demographic and infectious disease models of buffaloes and other domestic hosts for animal trypanosomosis (surra) caused by Trypanosoma evansi were developed. The animal models contained deterministic and stochastic elements and were linked to simulate the benefit of control regimes for surra in village domestic animal populations in Mindanao, Philippines. The impact of the disease on host fertility and mortality were key factors in determining the economic losses and net-benefit from the control regimes. If using a high (99%) efficacy drug in surra-moderate to high risk areas, then treating all animals twice each year yielded low prevalence in 2 years; targeted treatment of clinically sick animals, constantly monitored (monthly), required 75% fewer treatments but took longer to reach a low prevalence than treating all animals twice each year. At high drug efficacy both of these treatment strategies increased the benefit over untreated animals by 81%. If drug efficacy declined then the benefit obtained from twice yearly treatment of all animals declined rapidly compared with regular monitoring and targeting treatment to clinically sick animals. The current control regimen applied in the Philippines of annual sero-testing for surra and only treating sero-positive animals provided the lowest net-benefit of all the control options simulated and would not be regarded as effective control. The total net-benefit from effective surra control for a typical village in a moderate/high risk area was 7.9 million pesos per annum (US $158,000). The value added to buffaloes, cattle, horses, goats/sheep and pigs as a result of this control was US $88, $84, $151, $7, $114 per animal/year, respectively.

A comparison of trapping methods for Tabanidae (Diptera) in North Queensland, Australia.

The ability to monitor the abundance and diversity of tabanid flies over wide areas requires effective and low-cost surveillance methods. Such monitoring activities help to quantify the risk of transmission of pathogens by tabanids. Here we examine the effectiveness and practicality of two types of trap (canopy traps and Nzi traps) and two types of attractant (octenol and carbon dioxide) for monitoring tabanid flies in tropical Australia. The Nzi trap consistently caught more tabanids and more species of tabanids than the canopy trap. It was also more robust and therefore required less maintenance in remote locations. The use of attractants substantially increased capture rates, both of individuals and species, and traps using both attractants were consistently the most effective. However, in remote locations, where it is not possible to check traps frequently, the use of attractants may not be feasible. When attractants were not used, the canopy trap caught very few tabanids, but the Nzi trap remained effective enough to be useful as a monitoring device. In addition, the number of tabanid species caught by the Nzi traps remained high, and included those that were most abundant. We therefore conclude that, in this region, Nzi traps are preferable for tabanid monitoring and that attractants greatly improve their effectiveness. However, for longterm monitoring, especially in remote locations, Nzi traps without attractants are a satisfactory option.

Genetic variability of Trypanosoma evansi isolates detected by inter-simple sequence repeat anchored-PCR and microsatellite.

Studies on genetic variability in Trypanosoma evansi have been limited by a lack of high-resolution techniques. In this study, we have investigated the use of inter-simple sequence repeats (ISSR) and microsatellites in revealing polymorphism among T. evansi isolates. Twelve ISSR primers and five microsatellite loci were used to generate polymorphic bands and alleles, respectively, to investigate the genetic variability among T. evansi isolates from Africa and Asia. Seven of the twelve ISSR primers showed variability between isolates with a total of 71 fragments of which 49(69%) were polymorphic. Microsatellite analysis revealed a total of 60 alleles. On average the ISSR markers revealed a higher genetic diversity (23%) than microsatellites (21.1%). The two techniques showed a strong agreement of r=0.95 for Dice and r=0.91 for Jaccard indices in estimating the genetic distances between isolates. The distance UPGMA tree revealed two major clusters of T. evansi which correlate with the minicircle classification of subtype A and B. The cophenetic correlation coefficient between Dice and Jaccard based matrices were r=0.79 for microsatellites and r=0.73 for ISSR indicating a strong agreement between dendrograms. The results suggest that both ISSR and microsatellites markers are useful in detecting genetic variability within T. evansi.

Immunization with recombinant beta-tubulin from Trypanosoma evansi induced protection against T. evansi, T. equiperdum and T. b. brucei infection in mice.

The beta-tubulin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi beta-tubulin shows 100%, 99.8%, 99.1%, and 98.6% homology with T. equiperdum, T. b. brucei, T. cruzi and T. danilewskyi, respectively, but is diverse from that of T. cyclops, showing only 51.6% of homology. Recombinant beta-tubulin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant beta-tubulin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818 and T. b. brucei STIB 940, showing 83.3%, 70% and 76.7% protection, respectively. Serum collected from the rabbit immunized with recombinant beta-tubulin inhibited the growth of T. evansi, T. equiperdum and T. b. brucei in vitro. Serum from mice and rabbits immunized with recombinant beta-tubulin recognized only T. evansi beta-tubulin and not mouse beta-tubulin. The results of this study demonstrated that the recombinant T. evansi beta-tubulin is a potential candidate for the development of a vaccine to prevent animal trypanosomiasis caused by these three trypanosome species.

Exercise-associated hyponatremia, renal function, and nonsteroidal antiinflammatory drug use in an ultraendurance mountain run.

OBJECTIVE: To study biochemical parameters and renal function in runners completing a 60 km mountain run and to investigate the incidence of exercise-associated hyponatremia (EAH). To assess the effects of nonselective nonsteroidal antiinflammatory medication (NSAIDs) and cyclooxygenase-2 (COX-2) selective nonsteroidal antiinflammatory medication (COXIBs) on these parameters. DESIGN: Observational cohort study. SETTING: Kepler Challenge 60 km mountain run, Te Anau, New Zealand, December 2003. PARTICIPANTS: One hundred thirty-one of the 360 runners entered in the race were prospectively enrolled as volunteers on the day before the race. MAIN OUTCOME MEASURES: Subjects were weighed at race registration the day before the race and at the finish line. Blood was taken within 5 minutes of finishing and was analyzed for serum sodium, creatinine, urea, and potassium concentrations, and hematocrit. Participants were questioned about medication use in the 24 hours before and during the race (NSAIDs, COXIBs, other medications). RESULTS: Complete data sets were obtained on 123 runners. Five athletes were biochemically hyponatremic [(Na) 130-134 mM] and four were hypernatremic [(Na) 146-148 mM]. Hyponatremia was associated with a mean weight gain of 1.32 kg (range, -1.5 to 1.6 kg). Serum [Na] varied inversely with weight change. Estimated creatinine clearance did not vary with percent weight loss. Estimated creatinine clearance declined with increasing runner age. Sixty-five percent of runners did not use any medication, whereas 20% had used NSAIDs and 15% had taken COXIBs. There were no statistically significant differences between NSAID and COXIB users in any measured parameters or between all NSAID and COXIB users when compared with nonusers. CONCLUSIONS: Mild asymptomatic EAH was found to occur in 4% of the volunteer ultraendurance mountain runner study group and was associated with a mean weight gain of 1.32 kg (range, -1.5 to 1.6 kg) during the race. Seven percent gained weight but remained normonatremic, suggesting other compensatory mechanisms. Hypernatremia was found in 3% and was associated with a mean weight loss. Postrace serum sodium concentration varied inversely with percent weight change. Runners using any NSAID were more likely to become hyponatremic. Estimated creatinine clearance increased with increasing age. Elevated serum creatinine concentration at the end of the race returned to normal when remeasured the week after the race. Thirty-five percent of runners were found to use NSAIDs or COXIBs. The measures of weight change and of serum sodium, potassium, urea, and creatine concentration did not differ between NSAID and COXIB users or between all nonsteroidal antiinflammatory users and nonusers.

Characterization of Trypanosoma evansi type B.

A distinctive feature of Trypanosoma evansi is the possession of a kinetoplast that contains homogeneous DNA minicircles, but lacks DNA maxicircles. Two major sequence variants of the minicircle have been described and here we have sequenced the type B variant and designed a specific PCR test to distinguish it from type A. Further a test based on maxicircles to distinguish T. brucei brucei from T. evansi was designed and evaluated. Using the designed PCR tests, we detected three type B isolates from camel blood samples collected in northern Kenya, more than 20 years after the first isolation of type B. Comparison of minicircle sequences from all four type B isolates shows >96% identity within the group, and 50-60% identity to type A minicircles. Phylogenetic analysis based on minicircle sequences reveals two clusters, one comprising isolates of type A and one of type B, while random amplification of polymorphic DNA show slight polymorphic bands within type B. Most T. evansi isolates analysed were heterozygous at a repetitive coding locus (MORF2). All type B isolates had one genotype designated 3/5 based on the alleles present. Three camel isolates, which had homogenous type A minicircles, lacked the RoTat 1.2 gene, while another five isolates were T. b. brucei, based on the heterogeneity of their minicircles and presence of maxicircles as demonstrated by PCR amplification of the gene for cytochrome oxidase subunit 1. Our results confirm the existence of T. evansi type B isolates, T. b. brucei and existence of T. evansi type A without RoTat 1.2 gene in Kenyan isolates.