The Use of Raw Poultry Waste as Soil Amendment Under Field Conditions Caused a Loss of Bacterial Genetic Diversity Together with an Increment of Eutrophic Risk and Phytotoxic Effects.

Poultry waste has been used as fertilizer to avoid soil degradation caused by the long-term application of chemical fertilizer. However, few studies have evaluated field conditions where livestock wastes have been used for extended periods of time. In this study, physicochemical parameters, metabarcoding of the 16S rRNA gene, and ecotoxicity indexes were used for the characterization of chicken manure and poultry litter to examine the effect of their application to agricultural soils for 10 years. Poultry wastes showed high concentrations of nutrients and increased electrical conductivity leading to phytotoxic effects on seeds. The bacterial communities were dominated by typical members of the gastrointestinal tract, noting the presence of pathogenic bacteria. Soils subjected to poultry manure applications showed statistically higher values of total and extractable phosphorous, increasing the risk of eutrophication. Moreover, while the soil bacterial community remained dominated by the ones related to the biogeochemical cycles of nutrients and plant growth promotion, losses of alpha diversity were observed on treated soils. Altogether, our work would contribute to understand the effects of common local agricultural practices and support the adoption of the waste treatment process in compliance with environmental sustainability guidelines.

Sisal wool skin disease in Merino sheep in the Argentine Patagonia region: Identification and molecular diagnosis of Corynebacterium bovis from skin lesions.

BACKGROUND: Sisal wool condition is a skin disease affecting Merino sheep in the Argentine Patagonia region. Corynebacterium spp. isolates have previously been isolated from skin swabs from lesions, while specific identification of the bacteria involved has not been reported. HYPOTHESIS/OBJECTIVES: The aim of this work was to characterize the bacterial agent isolated from sisal wool lesions and to develop a diagnostic method for field surveillance. MATERIALS AND METHODS: Molecular identification of a collection of 72 isolates obtained previously was performed using PCR and 16S rRNA and rpoB sequencing. A field survey was carried out on two farms in the Río Negro province of Argentine Patagonia. Swab samples from sheep with and without skin lesions were collected and analysed by PCR and culture. RESULTS: Isolates analysed were confirmed by sequencing as Corynebacterium bovis. Using a PCR test without culture step, all field samples from affected sheep were positive for C. bovis; samples from the healthy skin from the same animals or clinically healthy sheep all were negative. CONCLUSIONS AND CLINICAL RELEVANCE: Sisal wool skin disease was associated with C. bovis infection based on culture and PCR methods; the latter may be useful for helping to pursue a disease control strategy.

A participatory surveillance of marsh deer (Blastocerus dichotomus) morbidity and mortality in Argentina: first results.

BACKGROUND: In an era of unprecedented socio-ecological changes, managing wildlife health demands high-quality data collection and the engagement of local communities. Blastocerus dichotomus, the largest South American deer, is Vulnerable to extinction mainly due to habitat loss. Diseases have been recognised as a potential threat, and winter mortality has been historically described in marsh deer populations from Argentina. Field difficulties have, however, prevented in-depth studies of their health status. RESULTS: Between May 2014 and April 2017, we investigated marsh deer morbidity and mortality in the two largest populations in Argentina. We collected data by means of a passive surveillance system that involved a network of researchers, field partners (veterinarians, park rangers, and local community), and decision makers. We sampled marsh deer during as well as outside mortality events. A total of 44 marsh deer with different body condition scores were evaluated. We obtained haematology and biochemistry values from animals with good body condition score. Marsh deer with poor body condition had a high burden of the ticks Amblyomma triste and Rhipicephalus microplus. Vector-borne agents such as Theileria cervi, Trypanosoma theileri, Trypanosoma evansi, Ehrlichia chaffeensis, Anaplasma platys, Anaplasma odocoilei, Anaplasma marginale, and Candidatus Anaplasma boleense were also found. Haemonchus spp., Ostertagia spp., and Trichostrongylus spp. were the most frequent gastrointestinal parasites in deer with poor body condition. A Multiple Correspondence Analysis reinforced a possible association of winter period with lower body score condition, high tick loads, infection with E. chaffeensis, and presence of harmful gastrointestinal parasites. CONCLUSIONS: Our approach allowed the establishment of a participatory surveillance network of marsh deer morbidity and mortality in Argentina. We report and analyse the first data obtained opportunistically within the framework of this network, providing information on the infectious and parasitic agents in marsh deer populations. The occurrence of Fasciola hepatica and Leptospira interrogans serovar pyrogenes is reported for the first time in wild marsh deer from Argentina. Our data will be useful to improve the interpretation of future mortality events. The field implementation of a surveillance network is key to a holistic approach to wildlife diseases.

Diversity of Empruthotrema Johnston and Tiegs, 1992 parasitizing batoids (Chondrichthyes: Rajiformes and Myliobatiformes) from the Southwest Atlantic Ocean, with description of three new species.

During an extensive research project involving 519 specimens of batoids, including 13 species of Rajiformes and Myliobatiformes (Chondrichthyes) from the Argentine Sea, three new species of Empruthotrema were found and are described using morphologic characteristics and two molecular markers: LSU rDNA and COI mtDNA. The new species can be distinguished from their congeners by the number and distribution of the marginal loculi, the length and morphology of male copulatory organ, and the presence of eyespots. Additionally, multivariate analysis identified the dimensions of the pharynx and ejaculatory bulb as diagnostic features. Host specificity and previous records of the genus in the region are discussed. This is the first description of new species in this genus for the Southwestern Atlantic Ocean, as well as for arhynchobatid hosts.

[Spotted fever in Argentina. Description of two clinical cases]. / Fiebre manchada en Argentina. Descripción de dos casos clínicos.

Rickettsioses are zoonotic tick-borne diseases. In Argentina, there are two epidemiological scenarios: jungle of Salta and Jujuy, involving vectors from the "Amblyomma cajennense Complex" (A. sculptum, and A. toneliae) and Rickettsia rickettsii as the main etiological agent; and the second scene to Delta del Rio Paraná and Samborombón Bay, where Amblyomma triste acts as a vector; and the provinces of Córdoba, La Rioja, San Luis and La Pampa where Amblyomma tigrinum is the vector. In this second scenario, Rickettsia parkeri is the causal agent. The spotted fever (SF) due to R. rickettsii is responsible for a severe cutaneous and systemic disease. Contrarily, R. parkeri produces benign and self-limited clinical manifestation. Here we describe a fatal SF case by R. rickettsii, in El Tunal, Salta and the first SF case due to R. parkeri in San Juan.

Morphological and molecular evidence for a new species of Pseudanisakis Layman & Borovkova, 1926 (Nematoda: Ascaridida), parasitizing Rajiformes in southern Southwest Atlantic waters.

Pseudanisakis argentinensis n. sp. is proposed to accommodate parasitic nematodes found in six skate species (Rajidae and Arhynchobatidae) examined from southern Southwest Atlantic waters. The new species differs from its congeners by the following combination of characters: a cupola on each lip, males with 8-12 pairs of precloacal genital papillae, a larger size for both males and females, a greater length-to-breadth ratio of the ventriculus and the presence of a small knob on the tip of the tail. Allometric growth was observed for several morphometric features; however, the slopes of the allometric relationships across host species exhibited non-significant differences and were considered as a strong evidence for conspecificity. Congruent results were obtained after the genetic characterization of the mitochondrial cytochrome c oxidase subunit 1 gene of worms obtained from different skate species, whose values of genetic divergence (1.3) lay within the range of intraspecific variation. Previous records of specimens referred to as Pseudanisakis tricupola in skates from South American waters are regarded as conspecific with P. argentinensis n. sp.

Transcriptome profiling of Diachasmimorpha longicaudata towards useful molecular tools for population management.

BACKGROUND: Diachasmimorpha longicaudata (Hymenoptera: Braconidae) is a solitary parasitoid of Tephritidae (Diptera) fruit flies of economic importance currently being mass-reared in bio-factories and successfully used worldwide. A peculiar biological aspect of Hymenoptera is its haplo-diploid life cycle, where females (diploid) develop from fertilized eggs and males (haploid) from unfertilized eggs. Diploid males were described in many species and recently evidenced in D. longicaudata by mean of inbreeding studies. Sex determination in this parasitoid is based on the Complementary Sex Determination (CSD) system, with alleles from at least one locus involved in early steps of this pathway. Since limited information is available about genetics of this parasitoid species, a deeper analysis on D. longicaudata's genomics is required to provide molecular tools for achieving a more cost effective production under artificial rearing conditions. RESULTS: We report here the first transcriptome analysis of male-larvae, adult females and adult males of D. longicaudata using 454-pyrosequencing. A total of 469766 reads were analyzed and 8483 high-quality isotigs were assembled. After functional annotation, a total of 51686 unigenes were produced, from which, 7021 isotigs and 20227 singletons had at least one BLAST hit against the NCBI non-redundant protein database. A preliminary comparison of adult female and male evidenced that 98 transcripts showed differential expression profiles, with at least a 10-fold difference. Among the functionally annotated transcripts we detected four sequences potentially involved in sex determination and three homologues to two known genes involved in the sex determination cascade. Finally, a total of 4674SimpleSequence Repeats (SSRs) were in silico identified and characterized. CONCLUSION: The information obtained here will significantly contribute to the development of D. longicaudata functional genomics, genetics and population-based genome studies. Thousands of new microsatellite markers were identified as toolkits for population genetics analysis. The transcriptome characterized here is the starting point to elucidate the molecular bases of the sex determination mechanism in this species.

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction.

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5' ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct.

Immunogenicity of hypothetical highly conserved proteins as novel antigens in Anaplasma marginale.

Anaplasma marginale is a tick-transmitted Gram-negative intraerythrocytic bacterium and the etiological agent of bovine Anaplasmosis. Even though considerable research efforts have been undertaken, Anaplasmosis vaccine development remains a challenging field. Outer-membrane-specific antigens responsible for the ability of more complex immunogens could have a significant role in the protective response. Thus, the identification of outer-membrane antigens represents a major goal in the development of bacterial vaccines. Considering that 40 % of the annotated proteins in A. marginale remain as hypothetical, we selected three candidate antigens, AM1108, AM127, and AM216 based on experimental evidence, in silico structure prediction of ß-barrel outer membrane, and orthology clustering. Sequence alignment and analysis demonstrated a high degree of conservation for the three proteins between the isolates from Argentina compared to the American strains. We confirmed the transcription of the three genes in the intraerythrocytic stage. AM1108 and AM216 recombinant proteins elicited specific T-cell response proliferation and a significant rise in TNF-α and IFN-γ transcript levels, respectively. Only AM1108 was able to be recognized by specific antibodies from infected bovines. This study allowed the identification of new candidate components of the outer-membrane fraction of A. marginale. Further studies will be required to analyze their potential as effective antigens for being included in rational vaccine strategies.

Genetic and morphological evidence reveals the existence of a new family, genus and species of Echinorhynchida (Acanthocephala).

Gymnorhadinorhynchus gen. n. is proposed to accommodate its type species, G. decapteri sp. n., a parasite of the marine fish Decapterus punctatus (Cuvier), caught from the coastal waters of Brazil. Gymnorhadinorhynchus decapteri sp. n. was morphologically most similar to species of two echinorhynchid families, the Rhadinorhynchidae and the Cavisomidae, particularly in the structure of the proboscis and the absence of somatic spines, respectively. This combination of morphological features made it difficult to assign our specimen to an extant family of the Acanthocephala. Therefore, in order to clarify the systematic placement of G. decapteri, a molecular phylogenetic analysis was performed based on the SSU and LSU rDNA and the mitochondrial cox1 gene sequences obtained for the new taxon and other 26 acanthocephalan species. The results of parsimony and maximum likelihood analyses, using individual, combined and concatenated sequence data, consistently indicate that the specimens do not belong to any known family of the Echinorhynchida. Rather, G. decapteri represents a distinct lineage that is closely related to the Transvenidae, but distantly related to both the Rhadinorhynchidae and the Cavisomidae. Gymnorhadinorhynchidae fam. n. is therefore erected. This newly described family can be distinguished from other families of Echinorhynchida by the combination of the following morphological characters: a proboscis cylindrical with 10 rows of 22-26 hooks, dorsoventral differences in proboscis hooks, basal hooks forming a ring and being abruptly larger than anterior hooks, absence of trunk spines and presence of four tubular cement glands. This combination, in addition to several molecular autapomorphies, justifies the erection of a new genus, Gymnorhadinorhynchus gen. n., in order to accommodate this new species.

Molecular Detection of Anaplasma marginale in Capybara (Hydrochoerus hydrochaeris) from Corrientes, Argentina.

Monitoring wildlife health is essential for understanding global disease patterns, particularly as vector-borne infections extend the geographic ranges and thereby hosts due to environmental shifts. Anaplasma marginale, primarily impacting cattle, has economic implications and has been found in diverse hosts, yet its presence in capybaras (Hydrochoerus hydrochaeris), influential in tick-borne pathogen spread, lacks comprehensive understanding. From 2015 to 2022, 14 capybaras were surveyed across two different areas of northeastern Argentina. In 1 of 14 (7%) capybaras, the presence of A. marginale was confirmed through the amplification of specific genes, msp5 and msp1ß. In addition, A. marginale DNA was detected in the capybara's blood sample through quantitative PCR, with a cycle threshold value of 30.81 (800 copies per reaction). Amplification of a fragment of the msp1α gene revealed PCR products of three different sizes, suggesting the presence of at least three coinfecting A. marginale variants in the capybara host. This study suggests that capybaras are wild hosts for A. marginale in the Ibera Wetlands in Argentina, potentially influencing the infection dynamics of both domestic and wild species. This finding highlights the necessity for thorough studies on the role of capybaras in disease dynamics, crucial for understanding wildlife health and the spread of disease.

Transovarial Transmission of Anaplasma marginale in Rhipicephalus (Boophilus) microplus Ticks Results in a Bottleneck for Strain Diversity.

Anaplasma marginale is an obligate intraerythrocytic bacterium of bovines, responsible for large economic losses worldwide. It is mainly transmitted by Rhipicephalus (Boophilus) microplus ticks and, despite mounting evidence suggesting transovarial transmission, the occurrence of this phenomenon remains controversial. We evaluated the vector competence of R. microplus larvae vertically infected with A. marginale to transmit the bacterium to a naïve bovine. A subgroup of engorged female ticks collected from an A. marginale-positive animal was dissected and the presence of the pathogen in its tissues was confirmed. A second subgroup of ticks was placed under controlled conditions for oviposition. After confirming the presence of A. marginale in the hatched larvae, an experimental infestation assay was conducted. Larvae were placed on an A. marginale-free splenectomized calf. The bacterium was detected in the experimentally infested bovine 22 days post-infestation. We analyzed the A. marginale diversity throughout the transmission cycle using the molecular marker MSP1a. Different genotypes were detected in the mammalian and arthropod hosts showing a reduction of strain diversity along the transmission process. Our results demonstrate the vertical transmission of A. marginale from R. microplus females to its larvae, their vector competence to transmit the pathogen, and a bottleneck in A. marginale strain diversity.

The Lambda Variant in Argentina: Analyzing the Evolution and Spread of SARS-CoV-2 Lineage C.37.

The second wave of COVID-19 occurred in South America in early 2021 and was mainly driven by Gamma and Lambda variants. In this study, we aimed to describe the emergence and local genomic diversity of the SARS-CoV-2 Lambda variant in Argentina, from its initial entry into the country until its detection ceased. Molecular surveillance was conducted on 9356 samples from Argentina between October 2020 and April 2022, and sequencing, phylogenetic, and phylogeographic analyses were performed. Our findings revealed that the Lambda variant was first detected in Argentina in January 2021 and steadily increased in frequency until it peaked in April 2021, with continued detection throughout the year. Phylodynamic analyses showed that at least 18 introductions of the Lambda variant into the country occurred, with nine of them having evidence of onward local transmission. The spatial--temporal reconstruction showed that Argentine clades were associated with Lambda sequences from Latin America and suggested an initial diversification in the Metropolitan Area of Buenos Aires before spreading to other regions in Argentina. Genetic analyses of genome sequences allowed us to describe the mutational patterns of the Argentine Lambda sequences and detect the emergence of rare mutations in an immunocompromised patient. Our study highlights the importance of genomic surveillance in identifying the introduction and geographical distribution of the SARS-CoV-2 Lambda variant, as well as in monitoring the emergence of mutations that could be involved in the evolutionary leaps that characterize variants of concern.

[Enteroparasitosis in a child population of the Hurlingham District, Buenos Aires]. / Enteroparasitosis en una población infantil del Municipio de Hurlingham, Buenos Aires.

INTRODUCTION: Intestinal parasites have the greatest impact on child populations as they can, in the long term, lead to stunted growth with advances in cognitive development. The transmission of these parasites is favored by environments of social vulnerability. METHODS: In a cross-sectional study carried out in the District of Hurlingham, in the suburbs of Buenos Aires, the prevalence of intestinal parasitoses and its associated risk factors in a child population attending Primary Health Care Centers was estimated. Coproparasitological survey, Graham test and a socio-environmental and labor survey were performed. RESULTS: A total of 130 children, 50.8% women and 49.2% men (0-12 years) were analyzed; 89.1% of those over 4 years of age were in school. In 28.5% of households, the mother or father had a formal job and 63.8% received the Universal Child Allowance. Conditions of social vulnerability were observed in the analyzed population: poor access to network water and sewage and environmental sanitation. The parasitological diagnosis revealed that 75 children [57.7% (95% CI: 48.7-66.3)] were parasitized. Protists were more frequent than helminths and the most prevalent species were Blastocystis spp, Enterobius vermicularis, Giardia lamblia and Cryptosporidium sp. The parasites in children were associated with schooling (p <0.01) and age (p <0.05). DISCUSSION: This work constitutes the first survey on intestinal parasitoses in the District of Hurlingham and the socioeconomic and environmental context where it is developed.

Introducción: Los parásitos intestinales tienen mayor impacto en las poblaciones infantiles ya que pueden, a largo plazo, conducir a un retraso en el crecimiento con alteración del desarrollo cognitivo. Sumado a esto, los entornos de vulnerabilidad social favorecen la transmisión y permanencia de estos parásitos en el ambiente. Métodos: En un estudio transversal realizado en el Municipio de Hurlingham (conurbano bonaerense) se estimó la prevalencia de enteroparasitosis y sus factores de riesgo asociados en una población infantil concurrente a Centros de Atención Primaria para la Salud locales mediante un análisis coproparasitológico, un test de Graham y una encuesta socioambiental y laboral. Resultados: Un total de 66 niñas (50.8%) y 64 niños (49.2%) entre 0 a 12 años fueron analizados. El 89.1% de los mayores de 4 años estaban escolarizados. En el 28.5% de los hogares la madre o el padre tenía trabajo formal y el 63.8% percibía la asignación universal por hijo (AUH). Se observaron condiciones de vulnerabilidad social en la población analizada, tales como: falta de acceso al agua de red y cloacas y un saneamiento ambiental deficiente. El diagnóstico parasitológico reveló un total de 75 casos [57.7% (IC 95%: 48.7-66.3)] de parasitosis. Los protistas fueron los parásitos más frecuentes y las especies más prevalentes: Blastocystis spp, Enterobius vermicularis, Giardia lamblia y Cryptosporidium sp. La presencia de parásitos estuvo asociada a la escolarización (p <0.01) y a la edad del niño (p <0.05). Discusión: Este fue el primer relevamiento sobre las enteroparasitosis en el municipio de Hurlingham y el contexto socioeconómico y ambiental donde se desarrollan.

Coxiella Endosymbiont of Rhipicephalus microplus Modulates Tick Physiology With a Major Impact in Blood Feeding Capacity.

In the past decade, metagenomics studies exploring tick microbiota have revealed widespread interactions between bacteria and arthropods, including symbiotic interactions. Functional studies showed that obligate endosymbionts contribute to tick biology, affecting reproductive fitness and molting. Understanding the molecular basis of the interaction between ticks and their mutualist endosymbionts may help to develop control methods based on microbiome manipulation. Previously, we showed that Rhipicephalus microplus larvae with reduced levels of Coxiella endosymbiont of R. microplus (CERM) were arrested at the metanymph life stage (partially engorged nymph) and did not molt into adults. In this study, we performed a transcriptomic differential analysis of the R. microplus metanymph in the presence and absence of its mutualist endosymbiont. The lack of CERM resulted in an altered expression profile of transcripts from several functional categories. Gene products such as DA-P36, protease inhibitors, metalloproteases, and evasins, which are involved in blood feeding capacity, were underexpressed in CERM-free metanymphs. Disregulation in genes related to extracellular matrix remodeling was also observed in the absence of the symbiont. Taken together, the observed alterations in gene expression may explain the blockage of development at the metanymph stage and reveal a novel physiological aspect of the symbiont-tick-vertebrate host interaction.

Integral approach for the evaluation of poultry manure, compost, and digestate: Amendment characterization, mineralization, and effects on soil and intensive crops.

The egg industry has increased its production worldwide during the last decades. Several waste management strategies have been proposed to treat large volumes of poultry manure. Composting and anaerobic digestion are the main stabilization processes used. However, there are disagreements on the criteria for applying raw and treated poultry manure to the soil. We studied the relationship between physicochemical, toxicological, microbiological, parasitological, and metabarcoding parameters of raw and treated poultry manure (compost and digestate). Subsequently, we evaluated the mineralization of C, N and P, and the effects of amended soil on horticultural and ornamental crops. Compost and digestate presented better general conditions than poultry manure for use as organic soil amendments. The highest pathogenic microorganism content (total and fecal coliforms, Escherichia coli, and Salmonella spp.) was recorded for poultry manure. Multivariate analyses allowed associating a lower phytotoxicity with compost and a higher microbial diversity with digestate. Therefore, only compost presented stability and maturity conditions. We found high released CO2-C, N loss, and P accumulation in soil amended with a high dose of poultry manure during mineralization. However, high doses of poultry manure and digestate increased the biomass production in the valorization assay. We recommend the soil application of stabilized and mature poultry manure-derived amendments, which reduce the negative impacts on the environment and promote more sustainable practices in agricultural systems.

Geography as non-genetic modulation factor of chicken cecal microbiota.

The gastrointestinal tract of chickens harbors a highly diverse microbiota contributing not only to nutrition, but also to the physiological development of the gastrointestinal tract. Microbiota composition depends on many factors such as the portion of the intestine as well as the diet, age, genotype, or geographical origin of birds. The aim of the present study was to demonstrate the influence of the geographical location over the cecal microbiota from broilers. We used metabarcoding sequencing datasets of the 16S rRNA gene publicly available to compare the composition of the Argentine microbiota against the microbiota of broilers from another seven countries (Germany, Australia, Croatia, Slovenia, United States of America, Hungary, and Malaysia). Geographical location played a dominant role in shaping chicken gut microbiota (Adonis R2 = 0.6325, P = 0.001; Mantel statistic r = 0.1524, P = 4e-04) over any other evaluated factor. The geographical origin particularly affected the relative abundance of the families Bacteroidaceae, Lactobacillaceae, Lachnospiraceae, Ruminococcaceae, and Clostridiaceae. Because of the evident divergence of microbiota among countries we coined the term "local microbiota" as convergent feature that conflates non-genetic factors, in the perspective of human-environmental geography. Local microbiota should be taken into consideration as a native overall threshold value for further appraisals when testing the production performance and performing correlation analysis of gut microbiota modulation against different kind of diet and/or management approaches. In this regard, we described the Argentine poultry cecal microbiota by means of samples both from experimental trials and commercial farms. Likewise, we were able to identify a core microbiota composed of 65 operational taxonomic units assigned to seven phyla and 38 families, with the four most abundant taxa belonging to Bacteroides genus, Rikenellaceae family, Clostridiales order, and Ruminococcaceae family.

Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis.

Bovine babesiosis caused by Babesia bigemina and B. bovis is an economically relevant tick-borne disease distributed over tropical and subtropical world regions. Animals that recover from the clinical disease can remain persistently infected, and those carriers are epidemiologically relevant since they can act as a source of infection to other animals through the tick bite. According to the manual of the World Organisation for Animal Health (OIE), the recommended molecular diagnosis test for both parasites is a nested polymerase chain reaction (nPCR) based on an amplification of a fragment of the rap-1 gene. Since nPCRs are time consuming, have a higher cost and risk of contamination, we propose a single step PCR for B. bigemina (BbiVESA) and B. bovis (BboVESA) based on the amplification of the multi-copy ves-1α gene. We developed these methods and we achieved a detection limit of 1 × 10-12 % parasitemia for B. bigemina and of 1 × 10-6 % for B. bovis using reference strains, which compared to the reference OIE tests, results in an improvement in sensitivity of six orders for B. bigemina. Finally, we tested 48 field samples from a babesiosis enzootic region where we were able to detect a higher proportion of positive animals with both VESA methods than with the reference rap-1 nPCRs. This difference was statistically significant for each Babesia species. Concordance between both diagnostic schemes based on Cohen's kappa coefficient showed minimal to non-agreement (κ = 0.32) for B. bigemina and non-agreement (κ = 0.16) for B. bovis since BbiVESA and BboVESA PCR tests showed a significantly higher detection capacity. In conclusion, the high sensitivity of the assay, together with the lower demand of time and reagents make the VESA PCR methods developed here a valuable diagnostic tool for the molecular detection and epidemiological survey of both Babesia pathogens.

The key to egress? Babesia bovis perforin-like protein 1 (PLP1) with hemolytic capacity is required for blood stage replication and is involved in the exit of the parasite from the host cell.

Bovine babesiosis is a tick-borne disease caused by apicomplexan parasites of the Babesia genus that represents a major constraint to livestock production worldwide. Currently available vaccines are based on live parasites which have archetypal limitations. Our goal is to identify candidate antigens so that new and effective vaccines against Babesia may be developed. The perforin-like protein (PLP) family has been identified as a key player in cell traversal and egress in related apicomplexans and it was also identified in Babesia, but its function in this parasite remains unknown. The aim of this work was to define the PLP family in Babesia and functionally characterize PLP1, a representative member of the family in Babesia bovis. Bioinformatic analyses demonstrate a variable number of plp genes (four to eight) in the genomes of six different Babesia spp. and conservation of the family members at the secondary and tertiary structure levels. We demonstrate here that Babesia PLPs contain the critical domains present in other apicomplexan PLPs to display the lytic capacity. We then focused on the functional characterization of PLP1 of B. bovis, both in vitro and in vivo. PLP1 is expressed and exposed to the host immune system during infection and has high hemolytic capacity under a wide range of conditions in vitro. A B. bovis plp1 knockout line displayed a decreased growth rate in vitro compared with the wild type strain and a peculiar phenotype consisting of multiple parasites within a single red blood cell, although at low frequency. This phenotype suggests that the lack of PLP1 has a negative impact on the mechanism of egression of the parasite and, therefore, on its capacity to proliferate. It is possible that PLP1 is associated with other proteins in the processes of invasion and egress, which were found to have redundant mechanisms in related apicomplexans. Future work will be focused on unravelling the network of proteins involved in these essential parasite functions.

Closing the Gaps to Understand the Tick Transmission of Anaplasma marginale among Giant Anteaters (Myrmecophaga tridactyla) in Argentina.

Anaplasma marginale, a well-known cattle pathogen of tropical and subtropical world regions, has been previously molecularly characterized in a giant anteater (Myrmecophaga tridactyla) from Corrientes, Argentina. Ticks or other hematophagous arthropod involved in the wild transmission cycle remained unknown. The aim of the present study was to analyze the simultaneous occurrence of A. marginale in blood samples and ticks from giant anteaters from Corrientes in order to investigate if ticks could be relevant in the transmission among these mammals. Blood samples from 50 giant anteaters collected in different years and 26 ticks Amblyomma dubitatum and A. sculptum were studied through the molecular amplification of two unequivocal species-specific genes from A. marginale: msp5 and msp1ß. Twenty five giant anteaters and tick organs (salivary glands, gut and oviduct) from 11 ticks tested positive to the A. marginale DNA amplification. The further molecular characterization through MSP1a tandem repeats analysis revealed the presence of genotypes circulating among giant anteaters that had been previously identified in cattle blood samples from the same geographical region. These results confirm the presence of A. marginale in giant anteaters in Corrientes and suggests that A. dubitatum and A. sculptum ticks could be involved in the transmission among giant anteaters. Future studies will determine the role of these tick species in the wild transmission cycle in the study area and the eventual connection with the domestic cycle.