Streptococcus equi subsp. zooepidemicus: High molecular diversity of Argentinian strains isolated from mares with endometritis.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a mucosal commensal of the lower genital tract in horses and is the most isolated bacterium causing endometritis in mares. The aim of this study was to determine the molecular diversity of S. zooepidemicus obtained from endometritis in mares in Buenos Aires province, Argentina. Thirty isolates obtained from the uterus of mares in 2005 and 2017 were studied. The MLST scheme was applied to identify the Argentinian genotypes and the clonal relationships and patterns of evolutionary descent were identified using the eBURST algorithm - goeBURST. Twenty six different Sequence types (STs) were identified, being only 11 of them previously reported in horses and also, from several host species and tissues. The other 15 STs were reported in Argentinian reproductive strains of mares in our study for the first time. The genotypes obtained from uterus in Argentina were not evenly distributed when all the published S. zooepidemicus STs were analysed, thus, it was not possible to establish that the same lineage circulates in our equine population. The fact that the identified genotypes were also reported in other countries, diverse samples and host species suggest that there is not a host, and an anatomical niche adaptation. Finally, the isolation of the same genotype in the vagina/clitoris and the uterus of the same mare highlights the versatility of S. zooepidemicus and its role as an opportunistic pathogen.

Comparative Degradome Analysis of the Bovine Piroplasmid Pathogens Babesia bovis and Theileria annulata.

Babesia bovis and Theileria annulata are tick-borne hemoprotozoans that impact bovine health and are responsible for considerable fatalities in tropical and subtropical regions around the world. Both pathogens infect the same vertebrate host, are closely related, and contain similar-sized genomes; however, they differ in invertebrate host specificity, absence vs. presence of a schizont stage, erythrocyte invasion mechanism, and transovarial vs. transstadial transmission. Phylogenetic analysis and bidirectional best hit (BBH) identified a similar number of aspartic, metallo, and threonine proteinases and nonproteinase homologs. In contrast, a considerably increased number of S54 serine rhomboid proteinases and S9 nonproteinase homologs were identified in B. bovis, whereas C1A cysteine proteinases and A1 aspartic nonproteinase homologs were found to be expanded in T. annulata. Furthermore, a single proteinase of families S8 (subtilisin-like protein) and C12 (ubiquitin carboxyl-terminal hydrolase), as well as four nonproteinase homologs, one with dual domains M23-M23 and three with S9-S9, were exclusively present in B. bovis. Finally, a pronounced difference in species-specific ancillary domains was observed between both species. We hypothesize that the observed degradome differences represent functional correlates of the dissimilar life history features of B. bovis and T. annulata. The presented improved classification of piroplasmid proteinases will facilitate an informed choice for future in-depth functional studies.

Novel Genotype of Streptococcus dysgalactiae subsp. equisimilis Associated with Mastitis in an Arabian Filly: Genomic Approaches and Phenotypic Properties.

Streptococcus dysgalactiae subsp. equisimilis (Sde) is a commensal bacterium of horses that causes opportunistic infections. The aim of the work was to study genotypic and phenotypic properties of the Sde strain related to equine neonatal mastitis. Sde was isolated from an 8 day-old filly and sequenced for genome analysis, antibiotic susceptibility tests and virulence factor (VF) assays. The Sde strain presented the novel emm-subtype stC839.12 and the novel multilocus-sequence type ST-670, which belonged to a specific equine genotype group. Although no specific genotypic mechanisms related to antibiotic resistance were found, it presented genes encoding efflux pumps and transporters pmrA, bmrC and lmrP. Genes encoding several putative VFs including emm, cpa, fbp-2, adcA, hyl, htrA, tig, slo, and ndk and loci-encoding phosphoenolpyruvate-protein phosphotransferase systems were identified. This is the first report of an equine neonatal mastitis case caused by a novel genotype and horse specific Sde strain.

Driving multisectoral antimicrobial resistance action in South America: Lessons learned from implementing an enhanced tripartite AMR country self-assessment tool.

As part of an innovative Tripartite-EU collaboration Project that supports seven South American countries, a Landscape Analysis Tool (LAT) was developed and implemented to collect data to complement the Tripartite AMR Country Self-Assessment Survey (TrACSS) process. The LAT enables collection of broader and deeper information to guide development of priority One Health activities, and strengthen national action plans to combat antimicrobial resistance. The Project developed the tool, trained a consultant pool in its use, and implemented it in conjunction with multi-sectoral country teams. The main results were seven priority-informed country workplans that proposed specific activities in line with the Strategic Objectives of each country's national action plan. LAT implementation clearly showed that the tool is a strong complement to the TrACSS process and that there can be considerable benefit to the process of collecting additional data layers, especially to strengthen country ownership of AMR-related information and solidifying multisectoral engagement. Countries elsewhere might consider implementing this complementary tool - either once to establish a baseline - or periodically to gain a better ongoing understanding of the situation on the ground.

Novel seM-types of Streptococcus equi subsp. equi identified in isolates circulating in Argentina.

BACKGROUND: Strangles is a worldwide infectious disease caused by Streptococcus equi subsp. equi that affects the upper respiratory tract of horses. Streptococcus equi subsp. equi characterisation by seM-typing is internationally used for epidemiological studies and comparison of isolates. OBJECTIVES: To identify and to compare the seM-types of Argentinian isolates of Streptococcus equi subsp. equi. STUDY DESIGN: Investigation of bacterial isolates using molecular and phylogenetic approaches. METHODS: A total of 59 Argentinian isolates of Streptococcus equi subsp. equi obtained between 2007 and 2019 were studied by seM-typing. The sequence similarity of Argentinian seM-types and the other alleles available on the seM database was determined using BLAST and phylogenetic analysis was performed using the Neighbour-Joining algorithm. The amino acid sequences were predicted and compared with the predicted amino acid sequence of the reference strain 4047 using the MEGA 7 software and PROVEAN tool. RESULTS: Eight seM-types were found among the isolates. Only one of them (seM-61) has been previously reported and the other seven alleles (seM-129, seM-130, seM-131, seM-132, seM-133, seM-134 and seM-135) were novel seM sequences. High genetic similarity was observed among the Argentinian seM-types, with the exception of seM-130. No functional effects of amino acid differences were predicted. MAIN LIMITATIONS: The number of related and unrelated isolates per year. CONCLUSIONS: Seven novel seM-types and seM-61 that were previously reported in Brazil were circulating in Argentina which were identified as circulating in Argentinian horses between 2007 and 2019. The high genetic similarity among the Argentinian and Brazilian seM-types suggests that there is a geographical distribution of strain types. The geographical restriction of strains is likely to reflect the movement of horses between different equine disciplines and neighbouring countries.

Serological survey suggests circulation of coronavirus on wild Suina from Argentina, 2014-2017.

Swine coronaviruses affecting pigs have been studied sporadically in wildlife. In Argentina, epidemiological surveillance of TGEV/PRCV is conducted only in domestic pigs. The aim was to assess the prevalence of TGEV/PRCV in wild Suina. Antibodies against these diseases in wild boar and captive collared peccary were surveyed by ELISA. Antibodies against TGEV were found in three collared peccaries (n = 87). No TGEV/PRCV antibodies were detected in wild boar (n = 160). Preventive measures should be conducted in contact nodes where the transmission of agents may increase. Epidemiological surveillance in wildlife populations and in captive animals before their reintroduction should be attempted.

PCR detection of Dirofilaria immitis in Aedes aegypti and Culex pipiens from urban temperate Argentina.

Dirofilariasis, a mosquito-borne disease of dogs caused by the nematode Dirofilaria immitis (Leidy; Spirurida: Onchocercidae), has now become a growing zoonotic concern. Based on direct microscopical observation, Aedes aegypti (L.) and Culex pipiens L. (Diptera: Culicidae) have been previously incriminated as potential vectors of D. immitis in urban temperate Argentina. In this study, an effort was made to provide evidence for this assumption by screening of mosquitoes for D. immitis infection using a polymerase chain reaction (PCR) assay. PCR primers were developed to specifically amplify the D. immitis-16S rRNA gene and to reliably detect 100th of the genomic equivalent (10 pg) of the infective third-stage larvae in mosquito pools of up to 30 individuals. Collection of mosquitoes was performed between September 2007 and April 2008 in premises known to be inhabited by D. immitis-infected dogs in Greater Buenos Aires. The final collection comprised 453 specimens belonging to 11 mosquito species of the genera Aedes, Culex, Ochlerotatus, and Psorophora. PCR assays were performed on 82 pools (n ≤ 20) of heads and abdomens separately, as this allows differentiating infective and non-infective stages of the parasite, respectively. Identification of the non-infective stage of D. immitis in A. aegypti and C. pipiens provided additional strong support of transmission of the parasite by these species. To our knowledge, this was the first PCR screening for D. immitis-infected mosquitoes in South America.

Multiresistant and blaCTX-M-14-Carrying Salmonella ser. Typhimurium Isolated During a Salmonellosis Outbreak in an Equine Hospital in Argentina.

Salmonella spp. causes digestive clinical signs in horses. Foals and hospitalized animals are more susceptible to the disease. Nowadays, the report of multidrug-resistant Salmonella spp. producer of extended-spectrum ß-lactamases, is more frequent. The aim of this work was to study the clonal relationship and antimicrobial susceptibility profiles among Salmonella ser. Typhimurium isolates, obtained during a salmonellosis outbreak in an Argentinian equine hospital. Thus, in 2017, we studied the genotypic profiles and the susceptibility to antimicrobials of the strains isolated from three animals with diarrhea in an equine hospital of Argentina. The pulsotype identified by pulsed-field gel electrophoresis was the same among the isolates. Also, this pulsotype had been previously detected in human and porcine isolates, suggesting the circulation of the same strains in different species. Multidrug-resistant isolates with different ß-lactam susceptibility profiles were identified and blaCTX-M-14 was detected for the first time from an isolate of equine-origin in Argentina. Salmonella ser. Typhimurium is an important pathogen in public and veterinary health, so our results emphasize the relevance of appropriate measures to prevent and control this disease. Furthermore, routine antibiotic susceptibility tests of local strains are needed to improve the empiric treatment of equine salmonellosis.

First molecular characterization of Babesia vogeli in two naturally infected dogs of Buenos Aires, Argentina.

Large piroplasms (>2.5microm) were detected by direct microscopical investigation in 34 out of 16,767 (0.20%) canine blood smears in the Southern region of Greater Buenos Aires. Genomic DNA was extracted from two parasitemic dogs and the hypervariable 18S RNA gene region of the pathogen was specifically amplified, sequenced, and aligned with corresponding gene sequences available in the GenBank. Phylogenetic trees were constructed and compared. 18S RNA gene sequences reliably segregated in three clearly distinguishable clades representing Babesia canis, Babesia vogeli and Babesia rossi isolates, respectively. The 18S RNA gene sequences of both Babesia isolates from Argentina affiliated to the B. vogeli branch. This finding represents the first molecular evidence of the existence of B. vogeli in Argentina.

Mitigated clinical disease in water buffaloes experimentally infected with Babesia bovis.

Water buffaloes (Bubalus bubalis) are raised in tropical and subtropical regions of the world, and act as hosts of Babesia bovis parasites and the tick vector Rhipicephalus microplus. As no clinical cases of B. bovis-infection have been reported, we hypothesized that, unlike bovines, water buffaloes respond asymptomatically to an acute infection. To test this hypothesis, we inoculated two groups of 24-month-old Mediterranean breed water buffaloes with 108 erythrocytes infected with two Argentine B. bovis isolates: BboM2P (n = 5) or BboS2P (n = 5). These strains displayed mild (BboM2P) or high (BboS2P) pathogenicity in Bos taurus calves of the same age (n = 5 and n = 1, respectively), when tested in parallel. In water buffaloes, no changes in body temperature were observed with both strains, and no hematocrit changes were detected in BboM2P-inoculated animals. In contrast, in the BboS2P-inoculated water buffalo group significant but relatively minor reductions in haematocrit values were noted compared to the infected bovine. The parasitemia attained in water buffaloes was considerably lower than in bovines and could only be detected by nested PCR, or indirectly via serology, whereas in most bovines, it could also be detected in Giemsa-stained smears under the light microscope. Our results show that water buffaloes present no or significantly mitigated clinical symptoms to B. bovis infections and suggest that they are able to substantially reduce and/or eliminate B. bovis parasites from circulation by an efficient innate immune mechanism.

Characterization of a papain-like cysteine protease essential for the survival of Babesia ovis merozoites.

Babesia ovis, a tick-transmitted intraerythrocytic protozoan parasite, causes severe infections in small ruminants from Southern Europe, Middle East, and Northern Africa. With the aim of finding potential targets for the development of control methods against this parasite, sequence analysis of its genome led to the identification of four putative cysteine proteases of the C1A family. Orthology between B. ovis, B. bovis, T. annulata, and T. parva sequences showed that each B. ovis C1A peptidase sequence clustered within one of the four ortholog groups previously reported for these piroplasmids. The ortholog of bovipain-2 of B. bovis and falcipain-2 of Plasmodium falciparum, respectively, was designated "ovipain-2" and further characterized. In silico analysis showed that ovipain-2 has the typical topology of papain-like cysteine peptidases and a highly similar predicted three dimensional structure to bovipain-2 and falcipain-2, suggesting susceptibility to similar inhibitors. Immunoblotting using antibodies raised against a recombinant form of ovipain-2 (r-ovipain-2) demonstrated expression of ovipain-2 in in vitro cultured B. ovis merozoites. By immunofluorescence, these antibodies reacted with merozoites and stained the cytoplasm of infected erythrocytes. This suggests that ovipain-2 is secreted by the parasite and could be involved in intra- and extracellular digestion of hemoglobin and/or cleavage of erythrocyte proteins facilitating parasite egress. A significant reduction in the percentage of parasitized erythrocytes was obtained upon incubation of B. ovis in vitro cultures with anti-r-ovipain-2 antibodies, indicating an important functional role for ovipain-2 in the intra erythrocytic development cycle of this parasite. Finally, studies of the reactivity of sera from B. ovis-positive and negative sheep against r-ovipain-2 showed that this protease is expressed in vivo, and can be recognized by host antibodies. The results of this study suggest that ovipain-2 constitutes a potential target for immunotherapies and drug development against ovine babesiosis.

[Aspergillus fumigatus in nasopharyngeal cavity of horses]. / Presencia de Aspergillus fumigatus en la cavidad nasofaríngea de equinos.

Aspergillus fumigatus is a cosmopolitan opportunistic fungal associated to rhinopharyngitis, sinusitis and guttural pouches infection with nasal discharges. All them are similar with Strangle's sign, the infectious disease produced by Streptococcus equi spp. The aim of this work was to detect A. fumigatus in healthy horses living in boxes and field. 226 nasopharyngeal swabbing samples were obtained by mycological routine. A. fumigatus was isolated in 26 (11.5%) horses.

Genome-wide analysis of peptidase content and expression in a virulent and attenuated Babesia bovis strain pair.

Identifying virulence determinants in Apicomplexan parasites remains a major gap in knowledge for members within this phylum. We hypothesized that peptidases would segregate with virulence between a virulent parent Babesia bovis strain and an attenuated daughter strain derived by rapid in vivo passage. Using the complete genome sequence of the virulent T2Bo strain, 66 peptidases were identified and active sites confirmed. The presence, sequence identity and expression levels were tested for each of the 66 peptidases in the virulent parent and attenuated daughter T2Bo strains using whole genome, targeted sequencing approaches and microarrays analyses. Quantitative PCR revealed that there was no significant difference in peptidase expression between the virulent and attenuated strains. We conclude that while peptidases may well play a required role in B. bovis pathogenesis, neither loss of peptidase gene content nor reduced gene expression underlies the loss of virulence associated with in vivo passage and attenuation.

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

BACKGROUND: Cysteine proteases have been shown to be highly relevant for Apicomplexan parasites. In the case of Babesia bovis, a tick-transmitted hemoparasite of cattle, inhibitors of these enzymes were shown to hamper intraerythrocytic replication of the parasite, underscoring their importance for survival. RESULTS: Four papain-like cysteine proteases were found to be encoded by the B. bovis genome using the MEROPS database. One of them, the ortholog of Plasmodium falciparum falcipain-2, here named bovipain-2, was further characterized. Bovipain-2 is encoded in B. bovis 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 B. bovis isolates from North and South America. Synteny studies demonstrated that the bovipain-2 gene has expanded in the genomes of two related piroplasmids, Theileria parva and T. annulata, into families of 6 and 7 clustered genes respectively. The bovipain-2 gene is transcribed in in vitro cultured intra-erythrocyte forms of a virulent and an attenuated B. bovis 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 in vitro cultured merozoites, as well as diffused in the cytoplasm of infected erythrocytes. Anti-bovipain-2 antibodies also reacted with B. bigemina-infected erythrocytes giving a similar pattern, which suggests cross-reactivity among these species. Antibodies in sera of two out of six B. bovis-experimentally infected bovines tested, reacted specifically with recombinant bovipain-2 in immunoblots, thus demonstrating expression and immunogenicity during bovine-infecting stages. CONCLUSIONS: Overall, we present the characterization of bovipain-2 and demonstrate its in vitro and in vivo 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.

Development of a tandem repeat-based multilocus typing system distinguishing Babesia bovis geographic isolates.

Mini- and microsatellite sequences have proven to be excellent tools for the differentiation of strains and populations in several protozoan parasites due to their high variability. In the present work we have searched the genome of the tick-transmitted bovine hemoprotozoon Babesia bovis for tandem repeats (TRs) that could be useful for a multilocus typing system. Hundred and nineteen sequences were shortlisted and tested in five common B. bovis reference isolates originating from distinct geographic locations of North and South America: Texas, USA (T2Bo), Mexico (RAD and Mo7), and Santa Fe and Salta, Argentina (R1A and S2P, respectively). Satellite sequences were PCR-amplified using specific primers, separated by polyacrylamide gel electrophoresis, visualized by silver staining and sized. Fourteen TR sequences could be reliably amplified in all isolates and displayed length polymorphism. All primers used were specific for B. bovis and did not amplify genomic DNA from the bovine host or from Babesia bigemina, the principal co-infecting bovine parasite in the Americas, allowing their future use in field surveys. The 14 satellite markers identified are distributed throughout the four chromosomes of B. bovis as follows: chromosome 1 (n=3), chromosome 2 (n=2), chromosome 3 (n=5), and chromosome 4 (n=4). Within the five B. bovis isolates we identified nine satellite marker loci with two alleles, three with three alleles, one with four and another with five alleles. In comparison to Theileria parva, a bovine hemoprotozoan that pertains to the same piroplasmida order and own a genome of similar size, the number of polymorphic TRs and the average number of alleles per TR locus seem to be significantly reduced in the B. bovis genome. Furthermore, the ratio of micro- to minisatellites in both B. bovis and T. parva is considerably lower than in other eukaryotes, as confirmed by bioinformatic analysis. The multilocus genotype of the five B. bovis isolates was assessed and the genetic distance between each other determined followed by cluster analysis based on neighbor joining. The resulting phenogram showed that B. bovis isolates segregated into three clusters according to their geographic origin. The presented marker system is suitable to explore various parameters of B. bovis populations such as genetic diversity, infection dynamics and their structure under different epidemiological situations, which are of crucial importance for improved control strategies.

Flow cytometry to evaluate Anaplasma marginale parasitemia using a fluorescent nucleic acid stain.

In this work we describe a flow cytometry-based method using SYTO16 (a DNA intercalating agent) to quantify Anaplasma marginale-infected erythrocytes in blood from bovine animals. The linearity and reproducibility of the results obtained with SYTO16 labeling followed by flow cytometry analysis make it a suitable approach for measurement of parasitemia in A. marginale infections.

Water Buffalos as carriers of Babesia bovis in Argentina.

The tick-transmitted hemoprotozoan Babesia bovis is a major causative agent of bovine babesiosis, an often fatal disease of cattle. The disease is widespread in the northeastern region of Argentina, where an increasing part of the livestock is composed of water buffalos. Although clinical cases of buffalo babesiosis have not been reported so far, the pathogen-transmitting tick vector has been occasionally observed by us to be feeding on water buffalos. We therefore set out to examine whether buffalos may constitute a reservoir of the parasite. Competitive enzyme-linked immunosorbent assay (cELISA) detected B. bovis-specific antibodies in 20% of investigated buffalos (21/103), while direct detection of the pathogen by nested PCR was demonstrated in 34% of the animals (35/103). In one field, more than 60% of investigated animals (22/36) tested positive by nested PCR. These results are discussed in the context of buffalo babesiosis reported in other countries and in view of the currently effected control measures against bovine babesiosis in the region.