Antileishmanial Drug Discovery and Development: Time to Reset the Model?

Leishmaniasis is a vector-borne parasitic disease caused by Leishmania species. The disease affects humans and animals, particularly dogs, provoking cutaneous, mucocutaneous, or visceral processes depending on the Leishmania sp. and the host immune response. No vaccine for humans is available, and the control relies mainly on chemotherapy. However, currently used drugs are old, some are toxic, and the safer presentations are largely unaffordable by the most severely affected human populations. Moreover, its efficacy has shortcomings, and it has been challenged by the growing reports of resistance and therapeutic failure. This manuscript presents an overview of the currently used drugs, the prevailing model to develop new antileishmanial drugs and its low efficiency, and the impact of deconstruction of the drug pipeline on the high failure rate of potential drugs. To improve the predictive value of preclinical research in the chemotherapy of leishmaniasis, several proposals are presented to circumvent critical hurdles-namely, lack of common goals of collaborative research, particularly in public-private partnership; fragmented efforts; use of inadequate surrogate models, especially for in vivo trials; shortcomings of target product profile (TPP) guides.

Molecular characterisation of five Sarcocystis species in domestic sheep (Ovis aries) from Spain.

The major aim of the present study was to determine by molecular methods whether the wide and narrow types of macroscopic sarcocysts in Spanish sheep belonged to different species, that is, Sarcocystis gigantea and Sarcocystis medusiformis, respectively. Additionally, we wanted to identify and characterize molecularly the species forming microscopic sarcocysts and determine the phylogenetic placement of all species found. Portions of the oesophagus, diaphragm and hind legs containing macroscopic sarcocysts were collected from slaughtered culled ewes at an abattoir in the Province of Madrid, Central Spain, but both macroscopic and microscopic sarcocysts were isolated for molecular examination. Genomic DNA from 63 sarcocysts (21 macroscopic, 42 microscopic) were examined at the cytochrome c oxidase subunit I gene (cox1), while selected isolates of each species found were further examined at the 18S and 28S ribosomal RNA (rRNA) genes. The 63 sarcocysts comprised five cox1 sequence types, each corresponding to a particular sarcocyst type, and thus represented five Sarcocystis spp. The slender fusiform and thick macrocysts belonged to S. medusiformis and S. gigantea, respectively. The microscopic sarcocysts belonged to Sarcocystis arieticanis, Sarcocystis tenella and a Sarcocystis mihoensis-like species with slanting thorn-like cyst wall protrusions, which was characterised molecularly for the first time. Based on its phylogenetic position, the S. mihoensis-like species probably uses corvids as definitive hosts.

Morphological and molecular characteristics of six Sarcocystis spp. from red deer (Cervus elaphus) in Spain, including Sarcocystis cervicanis and three new species.

Samples of muscle tissue from the diaphragm, oesophagus and/or heart of eight adult red deer (Cervus elaphus hispanicus) from the Quintos de Mora Park in Toledo, Central Spain, were screened for sarcocysts by means of the compression method. From positive samples, individual sarcocysts were excised and examined in wet mounts under a light microscope (LM) in order to study their basic morphology before being preserved for molecular studies. In all red deer examined, only microscopic sarcocysts were found. Those in the diaphragm and oesophagus were spindle-shaped and about 1 × 0.1 mm in size, while those in cardiac muscle were sac-like and 500-800 × 80-180 µm. By LM, the sarcocysts either had densely packed, about 8-µm-long, hair-like protrusions (type 1), sparsely distributed indistinct projections (fuzzy outline; type 2) or no visible protrusions (smooth surface; type 3). In cardiac muscle, only sarcocysts without visible protrusions were found. One of the latter sarcocysts was examined by scanning electron microscopy (SEM) and found to possess thin ribbon-like protrusions. Forty-eight sarcocysts isolated from the diaphragm, oesophagus and heart of one red deer, as well as 55 sarcocysts from the heart of three other red deer, 103 sarcocysts in total, were characterized molecularly through PCR amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of the mitochondrial genome, revealing the presence of six major cox1 sequence types. Each type comprised either a single sequence (three types) or a collection of several identical or nearly identical sequences. From selected isolates possessing each of these cox1 sequence types, the complete 18S ribosomal RNA (rRNA) gene was amplified and sequenced directly and/or after cloning of the 5' end half. Supported by the sequence data from the latter gene, as well as the morphology of the sarcocysts from which the sequences originated, the six cox1 sequence types were considered to represent six separate Sarcocystis spp. Two cox1 sequence types were identified as belonging to the previously characterized species Sarcocystis hjorti (one sequence/sarcocyst) and Sarcocystis linearis (38 sequences/sarcocysts), respectively, whereas four sequence types were new. One of the latter types was assigned to the previously named species Sarcocystis cervicanis from red deer, since this sequence type was obtained from 52 sarcocysts from cardiac muscle, which matched the original morphological description (smooth surface) and habitat of this species. The remaining three sequence types were assigned to the three new species Sarcocystis iberica (one sequence/sarcocyst) Sarcocystis venatoria (10 sequences/sarcocysts) and Sarcocystis morae (one sequence/sarcocyst), respectively. The two species S. iberica and S. venatoria shared the same sarcocyst morphology (type 1) and habitat (diaphragm) and had virtually identical 18S rRNA gene sequences, but differed by 4% at cox1, which was considered sufficient to regard them as separate species. The single sarcocyst of S. morae (from the oesophagus) examined by LM had a smooth wall and this species was therefore believed to have the same type of ribbon-like protrusions (ultrastructurally) as sarcocysts of S. cervicanis and S. linearis, which were also the species most closely related to S. morae at cox1. Thus, there seems to be three species with similar ribbon-like cyst wall protrusions in red deer (S. cervicanis, S. linearis, S. morae), as well as three species with similar hair-like protrusions (S. hjorti, S. iberica, S. venatoria). Sarcocysts of S. cervicanis were only identified in cardiac muscle, whereas sarcocysts of S. linearis were found mainly in the diaphragm and oesophagus and rarely in the heart. The relative number of cox1 haplotypes was greater among sequences/isolates of S. linearis (17/38) than among isolates of S. cervicanis (7/52). Four of the species examined (S. cervicanis, S. linearis, S. iberica, S. venatoria) possessed considerable intra-isolate (intra-genomic) sequence variation (insertions/deletions, substitutions) in the 18S rRNA gene.

Immunization against Lamb Haemonchosis with a Recombinant Somatic Antigen of Haemonchus contortus (rHcp26/23).

Haemonchosis, caused by the abomasal nematode Haemonchus contortus, is a common parasitic disease of sheep. Our previous results showed that a soluble fraction from adult stages of the nematode (p26/23) induced partial protection against challenge. Recombinant DNA technology was applied to obtain a synthetic protein (rHcp26/23). Immunological assays (ELISA, Western blotting, and immunolocalization), using sera from lambs immunized with p26/23, confirmed the identity of the recombinant protein and demonstrated that the synthetic protein is equivalent to the purified protein employed in the previous immunoprophylaxis studies. Vaccination of lambs with 300 mug of rHcp26/23 and Freund's adjuvant elicited a notable specific antibody response. Immunization did not induce any significant protection after challenge with 16000 infective larvae of H. contortus, and comparable values for parasite faecal egg output, packed cell volume, and abomasal parasite burdens were found in vaccinated and control animals.

Age distribution and seasonal dynamics of abomasal helminths in wild red deer from central Spain.

A study on age distribution and seasonal dynamics of abomasal helminths in wild red deer was conducted in central Spain, by monthly samplings of fawns (<1 yr), subadult (1-2 yr), and adult (>2 yr) animals. Both intensity and prevalence of abomasal parasitism were higher in older animals, particularly in males. A bimodal pattern for intensity of infection by gastrointestinal parasites was observed. Maximum values attained in winter and summer may be related to variation in climate and the shifting availability of forage resources. The pattern was largely due to the contribution of Spiculopteragia asymmetrica/Spiculopteragia quadrispiculata, whereas the other species found (Ostertagia leptospicularis/Ostertagia kolchida and Ostertagia drozdzi/Ostertagia ryjikovi) occurred with lower prevalence and intensity of infection. Among these ostertagiines, the ratio for major and minor morphotypes of males of respective species and the relative abundance of males and females were stable through the annual cycle.

Haemonchus contortus-sheep relationship: a review

La hemoncosis, causada por el nematodo del abomaso de rumiantes Haemonchus contortus, constituye una de las enfermedades parasitarias más notables del ganado ovino en todo el mundo. Las infecciones provocan síndromes anémicos y de mala digestión/absorción que pueden causar la muerte en los casos agudos y disminución de la producción en las formas crónicas. Las claves principales para la aparición de esta enfermedad son el comportamiento biológico del helmito, su patogenia, además de la respuesta del hospedador. El conocimiento actualizado de estos aspectos permitirá una mayor eficaciencia de los métodos de diagnóstico y contro del proceso y, como consecuencia, la disminución de los riesgos de aparición de esta enfermedad. La presente revisión analiza y discute algunos aspectos de la relación ganado ovino-H. contortus de interés para disminuir el impacto de esta parasitosis sobre la salud y producción ganaderas.

Haemonchosis, caused by the abomasal nematode Haemonchus contortus, is among the most relevant parasitic diseases of small ruminants all over the world. The infections are responsible for anemic and bad digestion/absorption syndromes causing the death of severely infected animals in acute cases and the reduction of production scores in the chronic forms of the disease. The keys for the appearance of the disease include the biological behavior of the helminth and mechanisms of pathogenesis, besides the host’s response. The updated knowledge of these aspects would result in a higher efficiency of diagnostic and control methods, thus reducing the risks of appearance of the disease. In the present review, several aspects of the sheep - H. contortus relationship are analyzed with the aim of reducing the impact of this parasitosis on livestock health and productivity.

Identical ITS-1 and ITS-2 sequences suggest Spiculopteragia asymmetrica and Spiculopteragia quadrispiculata (Nematoda: Trichostrongylidae) constitute morphologically distinct variants of a single species.

Sequences of ITS-1 and ITS-2 rDNA for adult males of Spiculopteragia asymmetrica and Spiculopteragia quadrispiculata in red deer (Cervus elaphus) were determined. They were found to be identical, suggesting that S. asymmetrica and S. quadrispiculata represent a single species and do not refute the concept of dimorphic species in the Spiculopteragia.