Release of lysosomal enzymes in Tetrahymena: a Ca²⁺-dependent secretory process

The ciliate Tetrahymena thermophila releases lysosomal enzymes into nutrient and starvation media. We show here that this process occurs selectively, i.e. without leakage of cytoplasmic components, as indicated by lack of release of isocitrate dehydrogenase, a cytosolic enzyme with high activity in Tetrahymena . The role of intracellular Ca²⁺ in the process was also investigated. The Ca²⁺ ionophore A23187 has strong stimulatory effects on this release. Ionophore stimulation is maximal in the presence of extracellular Ca²⁺ but can occur also in its absence. Quin 2 fluorescence measurements indicate that intracellular Ca²⁺ increases in both cases. Mg²⁺ completely prevents the stimulatory effects of A23187. Ionomycin, another Ca²⁺ ionophore, also stimulates lysosomal enzyme release with a maximal response in the presence of extracellular Ca²⁺ . Measurements of extracellular isocitrate dehydrogenase showed that ionophore-stimulated lysosomal enzyme release can take place without leakage of cytoplasmic components. The observations that divalent cation ionophores stimulate selective lysosomal enzyme release and that this effect is strongest in the presence of external Ca²⁺ indicate that this cation plays a crucial role in the control of this process in Tetrahymena . Together with other observations they support the view that a subpopulation of Tetrahymena lysosomes has properties like those of secretory vesicles.Centro de Estudios Parasitológicos y de Vectore

Rhipicephalus (Boophilus) Microplus Ticks can Complete their Life Cycle on the Water Buffalo (Bubalus bubalis)

Rhipicephalus (Boophilus) microplus is considered one of the most important ectoparasites of cattle worldwide. Due to the increase in the number of water buffaloes (Bubalus bubalis) in R. microplus-infested areas, this study was designed to determine whether these ruminants are able to sustain the complete tick life cycle. To this aim, a seven-month old water buffalo of the Mediterranean breed and a Holstein bovine of the same age, both tick-naïve, were infested with R. microplus tick larvae, and the parasitic and non-parasitic tick stages were analyzed and compared. The studied parameters include the number of recovered engorged females, the time points at which the first and last engorged females fell to the ground; the pre-oviposition duration, the percentage of hatching and the reproductive efficiency index. No statistically significant differences were found between the buffalo and the bovine in all parameters measured. It was concluded that the water buffalo can act as a suitable reservoir for R. microplus ticks. These results should be taken into account when implementing tick control and eradication campaigns in water buffalo grazing lands

Release of lysosomal enzymes in Tetrahymena: a Ca²⁺-dependent secretory process

The ciliate Tetrahymena thermophila releases lysosomal enzymes into nutrient and starvation media. We show here that this process occurs selectively, i.e. without leakage of cytoplasmic components, as indicated by lack of release of isocitrate dehydrogenase, a cytosolic enzyme with high activity in Tetrahymena . The role of intracellular Ca²⁺ in the process was also investigated. The Ca²⁺ ionophore A23187 has strong stimulatory effects on this release. Ionophore stimulation is maximal in the presence of extracellular Ca²⁺ but can occur also in its absence. Quin 2 fluorescence measurements indicate that intracellular Ca²⁺ increases in both cases. Mg²⁺ completely prevents the stimulatory effects of A23187. Ionomycin, another Ca²⁺ ionophore, also stimulates lysosomal enzyme release with a maximal response in the presence of extracellular Ca²⁺ . Measurements of extracellular isocitrate dehydrogenase showed that ionophore-stimulated lysosomal enzyme release can take place without leakage of cytoplasmic components. The observations that divalent cation ionophores stimulate selective lysosomal enzyme release and that this effect is strongest in the presence of external Ca²⁺ indicate that this cation plays a crucial role in the control of this process in Tetrahymena . Together with other observations they support the view that a subpopulation of Tetrahymena lysosomes has properties like those of secretory vesicles.Centro de Estudios Parasitológicos y de Vectore

Cysteine proteinase C1A paralog profiles correspond with phylogenetic lineages of pathogenic piroplasmids

Piroplasmid parasites comprising of Babesia, Theileria, and Cytauxzoon are transmitted by ticks to farm and pet animals and have a significant impact on livestock industries and animal health in tropical and subtropical regions worldwide. In addition, diverse Babesia spp. infect humans as opportunistic hosts. Molecular phylogeny has demonstrated at least six piroplasmid lineages exemplified by B. microti, B. duncani, C. felis, T. equi, Theileria sensu stricto (T. annulata, T. parva, and T. orientalis) and Babesia sensu stricto (B. bovis, B. bigemina, and B. ovis). C1A cysteine-proteinases (C1A-Cp) are papain-like enzymes implicated in pathogenic and vital steps of the parasite life cycle such as nutrition and host cell egress. An expansion of C1A-Cp of T. annulata and T. parva with respect to B. bovis and B. ovis was previously described. In the present work, C1A-Cp paralogs were identified in available genomes of species pertaining to each piroplasmid lineage. Phylogenetic analysis revealed eight C1A-Cp groups. The profile of C1A-Cp paralogs across these groups corroborates and defines the existence of six piroplasmid lineages. C. felis, T. equi and Theileria s.s. each showed characteristic expansions into extensive families of C1A-Cp paralogs in two of the eight groups. Underlying gene duplications have occurred as independent unique evolutionary events that allow distinguishing these three piroplasmid lineages. We hypothesize that C1A-Cp paralog families may be associated with the advent of the schizont stage. Differences in the invertebrate tick host specificity and/or mode of transmission in piroplasmid lineages might also be associated with the observed C1A-Cp paralog profiles

In Silico Survey and Characterization of Babesia microti Functional and Non-Functional Proteases

Human babesiosis caused by the intraerythrocytic apicomplexan Babesia microti is an expanding tick-borne zoonotic disease that may cause severe symptoms and death in elderly or immunocompromised individuals. In light of an increasing resistance of B. microti to drugs, there is a lack of therapeutic alternatives. Species-specific proteases are essential for parasite survival and possible chemotherapeutic targets. However, the repertoire of proteases in B. microti remains poorly investigated. Herein, we employed several combined bioinformatics tools and strategies to organize and identify genes encoding for the full repertoire of proteases in the B. microti genome. We identified 64 active proteases and 25 nonactive protease homologs. These proteases can be classified into cysteine (n = 28), serine (n = 21), threonine (n = 14), asparagine (n = 7), and metallopeptidases (n = 19), which, in turn, are assigned to a total of 38 peptidase families. Comparative studies between the repertoire of B. bovis and B. microti proteases revealed differences among sensu stricto and sensu lato Babesia parasites that reflect their distinct evolutionary history. Overall, this data may help direct future research towards our understanding of the biology and pathogenicity of Babesia parasites and to explore proteases as targets for developing novel therapeutic interventions.Instituto de PatobiologíaFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Florin-Christensen, Monica. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wieser, Sarah Nathaly. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina.Fil: Wieser, Sarah Nathaly. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Suarez, Carlos E. USDA-ARS. Animal Disease Research Unit; Estados UnidosFil: Suarez, Carlos E. Washington State University. Department of Veterinary Microbiology and Pathology; Estados UnidosFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Molecular detection of Sarcocystis aucheniae in the blood of llamas from Argentina

Sarcocystis aucheniae are apicomplexan protozoa that infect South American camelids (SACs), giving rise to macroscopic cysts similar to rice grains in skeletal muscles. Visual detection of macrocysts in slaughtered animals hampers commercialization of SAC meat, a highly relevant economic exploitation for Andean rural families. Importantly, the consumption of undercooked S. aucheniae-infested meat causes gastroenteritis. A carnivore definitive host, possibly the dog, acquires the parasite when feeding on infected SAC meat, and later eliminates infective oocysts in its feces. The parasite cycle is completed when SACs ingest contaminated water or pastures. We hypothesized that parasite DNA can be detected in SAC blood using molecular methods. In order to test this hypothesis, a seminested PCR format was specifically designed to target the hypervariable 18S rRNA gene region of S. aucheniae. PCR conditions were optimized using genomic DNA extracted from macrocyst bradyzoites. A detection limit of up to 1 parasite in 10 l of llama blood was established based on DNA samples extracted from aliquots of S. aucheniae bradyzoite-spiked non-infected llama blood. The seminested PCR allowed to detect natural infections of S. aucheniae in llama blood samples originating in the Andean flatlands of Argentina. Specific amplification of S. aucheniae DNA was corroborated by amplicon sequencing. This is the first report of S. aucheniae detection in llama blood, which provides a valuable diagnostic tool for epidemiological studies and for the evaluation of the efficacy of control measures for this parasitosis.Sarcocystis aucheniae es un protozoo apicomplexa que infecta a camélidos sudamericanos (CS), dando lugar a la formación de quistes macroscópicos similares a granos de arroz en los músculos esqueléticos. La detección visual de macroquistes en animales faenados dificulta la comercialización de la carne de CS, una explotación de gran relevancia para la economía de las familias rurales andinas. Es importante destacar que el consumo de carne infectada con S. aucheniae no suficientemente cocida causa gastroenteritis. Un hospedador definitivo carnívoro, posiblemente el perro, adquiere el parásito cuando se alimenta de carne de CS infectada y luego elimina ooquistes infectivos en las heces. El ciclo del parásito se completa cuando un CS ingiere agua o pasturas contaminadas. Hemos hipotetizado que es posible detectar ADN del parásito en la sangre de CS usando métodos moleculares. Para poner a prueba esta hipótesis, se dise˜nó una PCR semianidada que utiliza como blanco una región del gen 18S ARNr específica para S. aucheniae. Se optimizaron las condiciones de la PCR usando ADN genómico extraído de bradizoítos presentes en macroquistes. Se estableció un límite de detección de un parásito en 10 l de sangre de llama, basado en muestras de ADN extraído de alícuotas de sangre de llama no infectada a las que se agregaron cantidades conocidas de bradizoítos de S. aucheniae. Más aún, la PCR semianidada permitió la detección de infecciones naturales por este parásito en muestras de sangre de llama de la Puna argentina. La amplificación específica de ADN de S. aucheniae fue corroborada por secuenciación de los productos de amplificación. Este es el primer reporte de la detección de S. aucheniae en sangre de llama. Además, este estudio contribuye una herramienta diagnóstica valiosa para estudios epidemiológicos y para la evaluación de la efectividad de medidas de control para esta parasitosis.Fil: Decker Franco, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romero, Sandra Raquel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Investigación y Desarrollo Tecnológico para la Agricultura Familiar Región NOA; ArgentinaFil: Carletti, Tamara. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Martin, Mara Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Bovipain-2, the falcipain-2 ortholog, is expressed in intraerythrocytic stages of the tick-transmitted hemoparasite Babesia bovis

<p>Abstract</p> <p>Background</p> <p>Cysteine proteases have been shown to be highly relevant for Apicomplexan parasites. In the case of <it>Babesia bovis</it>, a tick-transmitted hemoparasite of cattle, inhibitors of these enzymes were shown to hamper intraerythrocytic replication of the parasite, underscoring their importance for survival.</p> <p>Results</p> <p>Four papain-like cysteine proteases were found to be encoded by the <it>B. bovis </it>genome using the MEROPS database. One of them, the ortholog of <it>Plasmodium falciparum </it>falcipain-2, here named bovipain-2, was further characterized. Bovipain-2 is encoded in <it>B. bovis </it>chromosome 4 by an ORF of 1.3 kb, has a predicted molecular weight of 42 kDa, and is hydrophilic with the exception of a transmembrane region. It has orthologs in several other apicomplexans, and its predicted amino acid sequence shows a high degree of conservation among several <it>B. bovis </it>isolates from North and South America. Synteny studies demonstrated that the <it>bovipain-2 </it>gene has expanded in the genomes of two related piroplasmids, <it>Theileria parva </it>and <it>T. annulata</it>, into families of 6 and 7 clustered genes respectively. The <it>bovipain-2 g</it>ene is transcribed in <it>in vitro </it>cultured intra-erythrocyte forms of a virulent and an attenuated <it>B. bovis </it>strain from Argentina, and has no introns, as shown by RT-PCR followed by sequencing. Antibodies against a recombinant form of bovipain-2 recognized two parasite protein bands of 34 and 26 kDa, which coincide with the predicted sizes of the pro-peptidase and mature peptidase, respectively. Immunofluorescence studies showed an intracellular localization of bovipain-2 in the middle-rear region of <it>in vitro </it>cultured merozoites, as well as diffused in the cytoplasm of infected erythrocytes. Anti-bovipain-2 antibodies also reacted with <it>B. bigemina</it>-infected erythrocytes giving a similar pattern, which suggests cross-reactivity among these species. Antibodies in sera of two out of six <it>B. bovis</it>-experimentally infected bovines tested, reacted specifically with recombinant bovipain-2 in immunoblots, thus demonstrating expression and immunogenicity during bovine-infecting stages.</p> <p>Conclusions</p> <p>Overall, we present the characterization of bovipain-2 and demonstrate its <it>in vitro </it>and <it>in vivo </it>expression in virulent and attenuated strains. Given the involvement of apicomplexan cysteine proteases in essential parasite functions, bovipain-2 constitutes a new vaccine candidate and potential drug target for bovine babesiosis.</p

Pursuing effective vaccines against cattle diseases caused by apicomplexan protozoa

Apicomplexan parasites are responsible for important livestock diseases that affect the production of much needed protein resources, and those transmissible to humans pose a public health risk. Vaccines, recognized as a cost-effective and environmentally friendly method for the prevention of infectious diseases in livestock, can avert losses in food production and decrease the exposure of humans to zoonotic pathogens. This review focuses on the need for and advances in vaccine development against the apicomplexan parasites Theileria spp., Babesia spp., Toxoplasma gondii, Neospora caninum, Eimeria spp., Besnoitia spp., Sarcocystis spp., and Cryptosporidium parvum. Together, the effect of these parasites on the cattle industry worldwide causes an enormous burden, yet they remain poorly controlled and very few effective and practical vaccines against them are available.Vaccine development is hampered by our scarce and limited knowledge of the biology and mechanisms of pathogenesis of these microorganisms, and the absence of correlates of host immune protection. More studies focused on these aspects as well as on the identification of parasite vulnerabilities that can be exploited for vaccine design are needed. Novel “omics” and gene editing approaches in understanding complex parasite biology together with advances in vaccinology will facilitate the development of effective, sustainable, and practical vaccines against cattle diseases caused by apicomplexan parasites. Such vaccines will help prevent animal and human diseases and allow production of enough animal protein to feed the growing human population in the twenty-first century and beyond

Unraveling the complexity of the rhomboid Serine protease 4 family of Babesia bovis using bioinformatics and experimental studies

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host–pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.The Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina; the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina; and the United States Department of Agriculture.https://www.mdpi.com/journal/pathogensdm2022Veterinary Tropical Disease