Soil fungi enable the control of gastrointestinal nematodes in wild bovidae captive in a zoological park: a 4-year trial.

The control of gastrointestinal nematodes among ruminants maintained in zoological parks remains difficult due to infective stages develop in the soil. For the purpose to improve the possibilities of the control of gastrointestinal nematodes (genera Trichostrongylus, Nematodirus, Chabertia and Haemonchus) affecting wild captive bovidae ruminants belonging to the subfamilies Antilopinae, Caprinae, Bovinae and Reduncinae, commercial pelleted feed enriched with a blend of 104-105 spores of both filamentous fungi Mucor circinelloides + Duddingtonia flagrans per kg meal was provided for a period of 3.5 years. All animals were dewormed at the beginning of the trial and also when exceeding a cut-off point of 300 eggs per gram of feces (EPG). The anthelmintic efficacy ranged between 96 and 100%. The need for repeating the administration of parasiticide treatment disappeared at the 24th month of study in the Antilopinae individuals, and at the 8th month in the Caprinae, Bovinae and Reduncinae. No side-effects were observed on the skin or in the digestive, respiratory or reproductive system. It was concluded that this strategy provides a sustainable tool for preventing the contamination of paddocks where captive ruminants are maintained, decreasing the risk of infection by gastrointestinal nematodes and consequently the need of frequent deworming.

Antigen characterization from second instars of oestrid bot flies for the detection of anti-Cephenemyia stimulator antibodies by ELISA in roe deer (Capreolus capreolus).

A study to determine the most appropriate antigen for use in the serodiagnosis of Cephenemyia (Diptera: Oestridae) infestation in roe deer (Capreolus capreolus) was carried out using immunoenzymatic tests. Serum samples from 43 roe deer from northern Spain were obtained post-mortem and corresponding numbers of bot fly larvae established. Three antigen complexes were tested, including Cephenemyia stimulator Clark excretory/secretory antigens (CsES), C. stimulator somatic antigens (CsSA) and Oestrus ovis L. (Diptera: Oestridae) excretory/secretory antigens (OoES). In addition, the composition of each antigen was analysed using an electrophoresis system. Cephenemyia stimulator larvae were found in 25% of roe deer; the mean intensity of infection was 24.3 larvae per infested animal. In the antigen analysis, CsSA showed four exclusive bands of molecular weight (17-19, 62, 65 and 67-70 kDa). A positive correlation between immunoglobulin G (IgG) values and total number of larvae was found with CsES and CsSA. The highest sensitivity value, negative predictive value and negative likelihood ratio were obtained using CsES. The highest specificity value, positive likelihood ratio and kappa value were achieved with CsSA. The predictive values of the enzyme-linked immunosorbent assay (ELISA) using CsES and CsSA reached statistical significance and seroprevalence values were 26-44%. The use of ELISA with CsES and CsSA seems promising in the non-invasive diagnosis of Cephenemyia infestation in roe deer.

Relationship between exposure to Fasciola hepatica in roe deer (Capreolus capreolus) and cattle extensively reared in an endemic area.

The aim of this work is to know the prevalence of Fasciola hepatica in 301 roe deer and in 676 beef cattle kept in an endemic area. Detection of antibodies was determined in roe deer using a homemade ELISA with excretory/secretory antigens (FhES) and a recombinant protein (FhrAPS). None of the deer passed eggs by faeces and none flukes in their livers were found. The seroprevalence of F. hepatica was 29% using FhES, with significantly higher values in the oldest ones (36%). Twenty-eight percent of the samples were positive to FhrAPS. Twenty-three percent of the cows eliminated eggs of F. hepatica and the seroprevalence was 67% using FhrAPS. No relationship between the seropositivity values of deer and cattle was demonstrated. The role of wild ruminants as reservoirs of F. hepatica is discussed. We encourage the use of ELISA to know the possibility of exposure to trematodes in wild ruminants.

The efficacy of four anthelmintics against Calicophoron daubneyi in naturally infected dairy cattle.

The paramphistomicidal activity of four anthelmintics in dairy cattle naturally infected by Calicophoron (Paramphistomum) daubneyi was evaluated. Seventy Friesian adult cows were treated at drying-off (19 albendazole; 23 netobimin; 13 closantel and 15 oxyclozanide), and 21 remained untreated as controls. The anthelmintic efficacy was determined by estimating the faecal egg count reduction (FECR) values for each of the anthelmintics. The reduction in the number of cows shedding eggs in the faeces was also estimated. The C. daubneyi egg-output was not fully suppressed following the administration of any of the parasiticides. The FECR values ranged from 0% to 26% in the cows receiving albendazole or netobimin, with 11-39% of cattle becoming negative after therapy. Better results were achieved with closantel and oxyclozanide, with FECR values of 97-99% and CPCR (cattle positive by coprology reduction) percentages of 85-93%. The observation of a similar efficacy with closantel and oxyclozanide against C. daubneyi led us to recommend the administration of closantel in those countries where oxyclozanide is not available.

Mixed production of filamentous fungal spores for preventing soil-transmitted helminth zoonoses: a preliminary analysis.

Helminth zoonoses are parasitic infections shared by humans and animals, being the soil-transmitted helminths (STHs) mainly caused by roundworms (ascarids) and hookworms. This study was aimed to assess the individual and/or mixed production of two helminth-antagonistic fungi, one ovicide (Mucor circinelloides) and other predator (Duddingtonia flagrans). Fungi were grown both in Petri plates and in a submerged culture (composed by water, NaCl, Na2HPO4 · 12 H2O, and wheat (Triticum aestivum)). A Fasciola hepatica recombinant protein (FhrAPS) was incorporated to the cultures to improve fungal production. All the cultured plates showed fungal growth, without difference in the development of the fungi when grown alone or mixed. High counts of Mucor spores were produced in liquid media cultures, and no significant differences were achieved regarding single or mixed cultures, or the incorporation of the FhrAPS. A significantly higher production of Duddingtonia spores after the incorporation of the FhrAPS was observed. When analyzing the parasiticide efficacy of the fungal mixture, viability of T. canis eggs reduced to 51%, and the numbers of third stage cyathostomin larvae reduced to 4%. It is concluded, the capability of a fungal mixture containing an ovicide (Mucor) and a predator species (Duddingtonia) for growing together in a submerged medium containing the FhrAPS offers a very interesting tool for preventing STHs.

Epidemiology, chronobiology and taxonomic updates of Rhinoestrus spp. infestation in horses of Sardinia Isle, Western Mediterranean (Italy).

From January to December 2008, 265 horses slaughtered in Sardinia (Italy) were examined for the presence of Rhinoestrus spp. (Diptera: Oestridae) through the examination of the nasal cavities and pharynges. Larvae were detected in 49% of the horses, with a mean intensity of infestation of 16.09 and abundance of 7.95. A total of 2108 larvae were collected, 66% of which were classified in first instar (L1), 22% in second instar (L2) and 12% in third instar (L3). The most frequent localization of larvae was the ethmoid, while the less one the larynx. According to the dynamics of Rhinoestrus larval stages, three periods in the chronobiology can be considered, the diapause (September-February) characterized by an absolute prevalence of first larval stage; the active phase of the endogenous phase (February-September) with an increase in the percentages of L2 and L3, and the exit phase (May-September), pointed by a further increase of L1. Morphological examination of L3 larvae revealed the presence of the Rhinoestrus purpureus features in 8% of the examined larvae, of 8% of the Rhinoestrus usbekistanicus features, while in 84% of the larvae were evidenced intermediate features. Contrastingly biomolecular analysis of the COI gene of the larvae evidenced uniformity at genetic level, confirming the presence of a unique species in the Mediterranean area. The results of the present paper, reveal the wide diffusion of rhinoestrosis among Sardinian horses, and suggest the need for applying appropriate control measures. Chemotherapy should be very useful if administered during the diapause period, for reducing the presence of L1 stages and interrupting thus the life cycle of this myiasis.

Infection by Paramphistomidae trematodes in cattle from two agricultural regions in NW Uruguay and NW Spain.

The analysis of infection by Paramphistomidae trematodes was conducted in two agricultural regions with different knowledge on this parasitosis. Faecal and blood samples were collected from 374 cattle in Salto (NW Uruguay) where there is a lack of information about paramphistomosis. A total of 429 cattle from Galicia (NW Spain), an area with previous records of infection by gastric flukes, were sampled. Diagnostics of trematodosis was developed by using a copromicroscopic probe and an ELISA with excretory/secretory antigens collected from adult Calicophoron daubneyi (Paramphistomidae) specimens. Results were evaluated according intrinsic and extrinsic factors. In the Uruguay, the percentage of cattle passing Paramphistomidae-eggs by faeces was 7% (95% Confidence Interval 5, 10). A significantly higher prevalence of paramphistomosis in the Hereford × Angus cattle (OR=3.5) was recorded, as observed for the oldest ruminants (>3.5 years). An overall seroprevalence of 29% (25, 34) was obtained by ELISA, with the highest values in the Friesians (OR=3), the youngest bovines (<2.5 years) and dairy cattle (Friesians). Twenty-six percent (22, 30) of the cattle from Spain passed eggs by faeces, and cattle aged 2.5-7 years reached significant highest prevalences. By means of the ELISA, a percentage of 55 cattle (50, 59) had antibodies against the gastric fluke, and the highest seroprevalence was observed among the bovines under 6 years. It is concluded that paramphistomosis is on the increase in cattle from NW Spain, partly due to the absence of an effective treatment against the trematode. There is a need for reducing the risk of infection by Paramhistomidae spp. in cattle from Uruguay, especially by improving their management to avoid exposure to the gastric trematode. Further studies are in progress for identifying the species of Paramphistomidae affecting ruminants in Uruguay.

Isolation of potentially useful antigens from cyathostomin third-stage larvae by using a fast protein liquid chromatography one-step method.

Three major protein complexes (51, 29, and 15 kDa, named P1 to P3, respectively) were resolved by gel filtration of the excretory/secretory antigens collected from a mixture of horse cyathostomin third-stage larvae (L3s). The potential application for the detection of infected horses was assessed with an enzyme-linked immunosorbent assay (ELISA) by the comparison of the serological and copromicroscopical results. The value of the area under the receiver operating characteristic (ROC) curve was higher than 0.9 when the three peaks were used. Elevated values (>90%) for the sensitivity, specificity, and the positive-likelihood ratio were also observed for all the antigen complexes. A significant increment in the IgG antibody levels 4 weeks prior to the observation of eggs in the feces of weanlings naturally infected was recorded. Our results indicate that the evaluation of chemotherapy is possible by using immunoenzymatic probes and fast protein liquid chromatography (FPLC)-purified antigens. Data collected in the present investigation indicate that FPLC isolation offers a very helpful one-step method for collecting antigens with diagnostic potential to be employed in immunoenzymatic probes.

A novel second instar Gasterophilus excretory/secretory antigen-based ELISA for the diagnosis of gasterophilosis in grazing horses.

We have developed a novel enzyme-linked immunosorbent assay (ELISA) based on excretory/secretory antigens of second instar Gasterophilus for the diagnosis of gasterophilosis in grazing horses. Between January 2007 and January 2009, two experiments were carried out on free-ranging horses in northwest Spain. During the first year, monthly blood samples were collected from a herd of 25 horses. In the second year, a monthly serological survey was conducted for a total of 398 different horses. All the sera were analyzed by ELISA using excretory/secretory antigens from Gasterophilus intestinalis (GphiL2ES) and Gasterophilus nasalis second-stage larvae (GphnL2ES). Climatic data were collected between January 2007 and January 2009 from local meteorological automated stations to establish the weather pattern in the study area. Observations of Gasterophilus eggs on the horses' hair and third instars passed in the faeces were also done. The kinetics of IgG response decreased against GphiL2ES from January to July, increased slowly from August and rose up to January. After a slight decrease in January, the absorbances against GphnL2ES reduced from April to August, when the lowest values were observed. The IgG values rose until the end of the study in January. Third instars were observed in the faeces in March to May, and Gasterophilus eggs were seen on the horses' hair from June to September. The highest IgG seroprevalences were achieved in winter (January-February; 100%) against both antigens. The lowest percentages of seropositivity were observed in June (3%) to the GphiL2ES, and in July (9%) to the GphnL2ES. The use of antigens from G. intestinalis second-stage larvae was shown to be suitable for diagnosing infestation by G. intestinalis or G. nasalis. We concluded that under oceanic climate conditions, the egg-laying period occurs from late spring, and eggs and first instars are found in the mouth in early summer. During summer the second instars move into the stomach and intestine, where the third-stage larvae remain until the end of winter, when pupation takes place. The adult horse bot fly emerges in the spring. Two treatments for the control of gasterophilosis are suggested: a curative in the summer to eliminate the first instars and a preventive in the autumn to suppress the second instars.

Application of the analysis of serum antibodies (immunoglobulins M and G) to estimate the seroprevalence of ovine oestrosis and to evaluate the effect of chemotherapy.

The analysis of immunoglobulin (Ig) M and IgG serum isotypes as indicators of the presence of Oestrus ovis developmental stages was carried out. A serological survey to discover the seasonal variations in the prevalence of oestrosis in sheep from an oceanic climate area was developed. Six hundred and sixty-nine blood samples were analyzed for the presence of IgM and IgG using an enzyme-linked immunosorbent assay and O. ovis second-stage larvae excretory/secretory antigens. In addition, the effect of an ivermectin-based treatment on the humoral immune response was measured. The percentage of positive animals was 54% for IgM and 55% for IgG. The highest percentages of sheep positive to IgM were recorded in the winter and to IgG in the summer. No animals positive to IgM were detected in the ivermectin-treated sheep. These results seem to underline that oestrosis is a parasitic disease to keep watch for in regions with an oceanic climate. A noninvasive evaluation of the success of chemotherapy seems possible by analyzing the serum IgM response.

The cost of intensified case finding and isoniazid preventive therapy for HIV-infected patients in Battambang, Cambodia.

SETTING: The current study evaluates one of four pilot sites initiated in Cambodia to establish feasible and effective ways to manage patients with human immunodeficiency virus (HIV) infection and tuberculosis (TB). OBJECTIVE: To measure the costs of intensified case finding (ICF) and isoniazid preventive therapy (IPT) services for HIV-infected patients in Battambang Province, Cambodia. DESIGN: We analyzed cost data retrospectively from September 2003 to February 2006 using a microcosting or ingredients-based approach and interviewed clinic personnel to determine the cost of ICF and IPT per person. RESULTS: Adherence to IPT at Battambang IPT clinic was high (86%) relative to other reported studies of IPT among HIV patients in developing countries. The estimated cost per TB case averted through ICF was US$363, while the estimated cost per TB case averted through IPT was US$955. CONCLUSION: Economic evaluations of TB-HIV integrated services are necessary as countries move to establish or scale-up these services. Based upon the estimated effectiveness of ICF and IPT used by other studies examining the provision of integrated HIV-TB services, the cost per TB case prevented by ICF and IPT in Battambang, Cambodia, is less than the reported cost of treating a new smear-positive TB case.

Hospital DOTS linkage in Indonesia: a model for DOTS expansion into government and private hospitals.

BACKGROUND: To accelerate DOTS expansion, the hospital sector and specialized chest clinics must be engaged. OBJECTIVE: To develop a model for public-private partnership through DOTS expansion into public and private hospitals in Indonesia. DESIGN: Model development included gaining commitment from stakeholders, stepwise training of hospital staff, and developing unified networks for case management, patient referral, laboratory quality assurance, supervision and evaluation. RESULTS: The number of notified tuberculosis (TB) cases (all forms and new smear-positive) increased dramatically from baseline. Together, hospitals and chest clinics accounted for a significant proportion of the total cases notified by the province (51% of total TB cases and 56% of new smear-positive cases in 2004). Compared to health centers and chest clinics, hospitals reported lower cure and success rates. Despite the option for referral to health centers, the majority of patients diagnosed in hospitals and chest clinics in 2002-2004 opted to be fully managed by the diagnosing facility. CONCLUSION: The roles and strengths of hospitals differ with regard to health centers, providing a rational basis for linkage of these health service components. In Yogyakarta, linkage became effective only after establishing a stakeholder-based provincial coordinating (DOTS) committee as the recognized interface between the National Tuberculosis Programme and various providers.

Evaluating the strengths and weaknesses of tuberculosis educational activities for prisoners in Honduras.

SETTING: Prisons throughout Honduras. OBJECTIVE: To assess the strengths, weaknesses, and opportunities to improve non-standardized tuberculosis (TB) educational campaign materials utilized in prisons. DESIGN: A qualitative assessment was conducted. Prison staff completed a survey describing development and implementation of educational activities at each prison (n=25). Completed surveys with photographs or copies of educational materials were collected and cataloged. The content, literacy demand, illustrations, learning stimulation, and cultural appropriateness of cataloged items were assessed using an adapted version of the Suitability Assessment of Materials Instrument. Results from this assessment were examined in conjunction with information collected through focus groups conducted with prisoners and interviews with prison staff. RESULTS: Campaign strengths included the development and dissemination of print materials appropriate for individuals with low literacy levels. By actively engaging prisoners in the campaigns, materials often incorporated the language, values, and experiences encountered within prison culture. Content of print materials and presentations focused on correcting misperceptions and facilitating acceptance of TB control activities. Campaign weaknesses included the creation of illustrations that could perpetuate the stigma associated with TB and use of some materials in which the purpose was not apparent. CONCLUSIONS: The campaigns were generally suitable, and the evaluation allowed for their improvement.

Synthesis and structural, biochemical, and pharmacological study of 3 beta-acyloxy-3 alpha-methoxycarbonyltropane derivatives.

A series of 3 beta-acyloxy-3 alpha-methoxycarbonyltropanes were synthesized and studied by 1H and 13C NMR spectroscopy, and the crystal structure of 3 alpha-methoxycarbonyl-3 beta-pyridincarbonyloxytropane (5d) was determined by X-ray diffraction. In CDCl3 solution, compounds 5a-f display the same preferred conformation. The pyrrolidine and piperidine rings adopt an envelope conformation flattened at N-8 and a distorted chair conformation puckered at N-8 and flattened at C3, respectively, with the N-substituent in the equatorial position with respect to the piperidine ring. The pharmacological profile of one of these compounds makes it an adequate candidate for the design of novel GABAB antagonist agents.

APHE-1 and APHE-2, two new antimicrobial and cytotoxic antibiotics. II. Physico-chemical properties and structure elucidation.

Two new pyrazolo-isoquinolinone antibiotics, APHE-1 and APHE-2, have been isolated from the culture filtrate and mycelia of Streptoverticillium griseocarneum NCIMB 40447. Molecular formulae were established as C13H12N2O for APHE-1 and C14H14N2O for APHE-2, by elemental analysis, NMR and mass spectra. 2D NMR techniques (1H-1H COSY-45 and 1H-13C correlated spectroscopy) have been applied to establish their structures.

Synthesis and structural, conformational, and pharmacological study of some of esters derived from 3-phenethyl-3-azabicyclo[3.2.1]octan-8-beta-ol and the corresponding N-endo-methyl quaternary derivatives.

A series of 8-beta-acyloxy-3-phenethyl-3-azabicyclo[3.2.1]octane and its N-endo methiodides were synthesized and studied by 1H and 13C NMR spectroscopy, and the crystal structure of 8-beta-p-chlorobenzoyloxy-3-phenethyl-3-azabicyclo[3.2.1]octane methiodide (2c) was determined by X-ray diffraction. In CDCl3 solution, 1b-1e display the same preferred conformation. The cyclopentane and piperidine rings adopt an envelope conformation flattened at C-8 and a distorted chair conformation puckered at C-8 and flattened at N-3, respectively, with the N-substituent in the equatorial position with respect to the piperidine ring. In all cases, methylation takes place from the endo position. The ability of the title compounds to antagonize the acetylcholine-induced contraction of guinea pig ileum is also reported. An initial structure-activity relationship is proposed.