Genetic Analysis of LigA, LipL32, Loa22, and OmpL1 Antigens from Leptospira spp. Sejroe Serogroup: How Close We Are To a Vaccine Against Bovine Leptospirosis?

Bovine leptospirosis has as main causative agents Leptospira spp. from Sejroe serogroup. Vaccination is a crucial step to control this infection. The use of conserved proteins among Leptospira spp. is of great importance for a protective immune response. The aim of the present study is to genetically analyze antigens of Leptospira spp. from Sejroe serogroup strains isolated from cattle for a preliminary evaluation of vaccine candidates. Genes associated with antigenicity-LigA, LipL32, Loa22, and OmpL1-were analyzed through bioinformatic and immunoinformatic tools. Despite high diversity observed in strains, on an amino acid level, highly conserved regions were observed (> 90%), particularly in LipL32 gene. Moreover, highly conserved amino acid regions (> 30 aa) were observed in all genes, regardless of species, geographical origin or biological source of isolation. Superposed structures of protein fragments including all the predicted MHC-II and B-Cell epitopes were demonstrated. Results presented herein are preliminary, but a fundamental step towards the development of an efficient vaccine against bovine leptospirosis, a silent but enormously concerning disease.

The same strain leading to different clinical outcomes: The enigma behind the canine leptospirosis.

Canine leptospirosis is a worldwide zoonosis, varying from asymptomatic and chronic infections to clinical acute disease. In many parts of the world Leptospira interrogans serogroup Icterohaemorrhagiae strains have great epidemiological importance, being the most prevalent on dogs. The present study aims to characterize and compare strains/sequences belonging to the serogroup Icterohaemorrhagiae recovered from clinically ill and asymptomatic dogs. Based on secY gene sequences of L. interrogans serovar Icterohaemorrhagiae, we have studied genetic diversity of strains obtained from 13 dogs, including dogs with clinical signs of acute leptospirosis, asymptomatic dogs and animals with chronic kidney disease, all of them from the same geographical area, the state of Rio de Janeiro, Brazil. No genetic variations on secY gene were observed between strains/groups. No significant associations were observed between clinical status and age, sex or vaccinal status. The same strain leads to different clinical outcomes on canine leptospirosis. The answer for this will rise from deep studies regarding whole genomic sequencing of the strains, as well as proteomics. Those studies may provide key information for understanding of the clinical manifestation of the disease.

Leptospira spp. strains associated with Bovine Genital Leptospirosis (BGL).

Bovine Genital Leptospirosis (BGL) is an important syndrome that leads to reproductive failures. The present study aimed to perform a molecular analysis of Leptospira spp. identified from genital and urine samples from in vivo naturally infected cows with poor reproductive performance. A total of 48 cows destined for culling due to low reproductive efficiency were selected and submitted to sampling. Uterine fragments, cervicovaginal mucus (CVM), and urine were collected from all of the cows and processed for culturing and PCR. One isolate was recovered from the uterus of one cow. Other 25 animals were PCR-positive, totaling 26 positive cows. Of them, 18 animals were positive in lipL32-PCR to genital samples, while only seven animals were positive in urine. From those, sequencing of secY gene was performed. Of the 21 good sequences obtained, 16 were L. interrogans, two were L. noguchii, two were L. santarosai and one was L. borgpetersenii. In order to evaluate the genetic similarity of sequences found herein and other sequences from bovines worldwide, a phylogenetic analysis and haplotype networks were performed. Cows with reproductive failures had a significant association (p < 0.05) with positive PCR of genital samples when compared to PCR of urine. None of the animals were positive for genital samples and urine simultaneously. A high diversity of leptospiral strains were found, even in animals of the same epidemiological region. Haplotype networks of L. interrogans showed clusters of sequences from the uterus and CVM with high similarity to other genital sequences originating from previous studies. L. borgpetersenii haplotype networks presented two major clusters with high similarity, even from worldwide sequences, while L. santarosai showed clusters with high genetic distances, even with all the sequences being from Brazil. This study reinforces the theory that BGL and renal infection are distinct diseases, as well as, genital samples are crucial for the diagnosis of cows with reproductive failures caused by leptospires. In addition, haplotype networks confirmed a high genetic similarity between sequences from the present study and Sejroe strains, reinforcing Sejroe strains as the main BGL agents.

Molecular epidemiology of Leptospira spp. serogroup Sejroe associated with chronic bovine leptospirosis.

Pathogenic bacteria Leptospira spp. are commonly associated with bovine leptospirosis, characterized chiefly by chronic and subclinical reproductive disorders. Strains from the Sejroe serogroup play a significant role in these chronic genital infections known as Bovine Genital Leptospirosis (BGL), which notably impact cattle health. This study aims to deepen our understanding of BGL by investigating the genetic diversity, geographical distribution, and specific anatomical sites of infection of the causative agents. Initially, uterine fragments and cervicovaginal mucus were collected from 47 cows and subjected to PCR targeting the lipL32 gene. Positive samples in lipL32-PCR (9 samples) underwent genotyping based on the secY gene. Subsequently, sequences were aligned with GenBank entries (108 sequences) and analyzed in silico. All nine sequences from this study were identified as L. interrogans with an identity >99 % to serogroup Sejroe reference strains (Norma and L53). In the broader analysis, the most prevalent species observed was L. borgpetersenii, followed by L. interrogans and L. santarosai. The haplotype network of L. interrogans revealed that haplogroups B and C exclusively included L. interrogans strains of genital origin, while haplogroup A encompassed strains from renal sources as well. These findings underscore the significance of the L. borgpetersenii genotype Hardjobovis and L. interrogans genotype Hardjoprajitno as the predominant circulating strains and highlight the existence of distinct haplogroups of pathogenic leptospires originating from genital sources. We advocate for the use of secY as an effective genetic marker for Leptospira spp. and stress the necessity for additional research prioritizing the genital tract. The outcomes of this study contribute to the development of improved control measures for chronic cattle diseases and provide valuable guidance for future investigations.

The presence of Leptospira spp. in the follicular fluid of naturally infected cows affects the overall efficiency of the in vitro embryo production technique.

The relationship between Leptospira infection and reproductive failures, as well as the mechanisms that lead to it, has not yet been fully established. It has been hypothesized that the presence of Leptospira spp. in the follicular fluid (FF) could impair the oocyte developmental competence. Thus, the impact of the presence of Leptospira spp. in the FF on in vitro embryo production (IVEP) outcomes was assessed. Dairy cows (n=244) from different farms were subjected to ovum pick-up for cumulus-oocyte complexes (COCs) collection. After PCR analysis of the FF, cows were retrospectively allocated into either: positive (POS-FF) or negative (NEG-FF) group. Statistical modeling was conducted using the farm, PCR result, and laboratory in which the IVEP was performed as effects. Noteworthy, 26.6% of the animals were positive for Leptospira spp., and 70% of farms had at least one POS-FF cow in the herd. POS-FF cows had a lower number of COCs recovered (22.6 ± 1.2 vs 15.0 ± 2.8, P=0.036), rate of viable COCs (85.6 ± 0.9% vs 78.1 ± 2.8%, P=0.015), number of good-quality COCs (16.0 ± 0.9 vs 9.8 ± 2.1, P=0.026), cleaved embryos (11.9 ± 0.7 vs 7.5 ± 1.5, P=0.032), and blastocysts (7.3 ± 0.4 vs 2.3 ± 0.7, P=0.044) yielded per cow. In conclusion, the presence of Leptospira spp. in the FF of naturally infected cows impaired the amount of COCs recovered, decreasing the overall IVEP efficiency.

Molecular Epidemiology of Pathogenic Leptospira spp. Infecting Dogs in Latin America.

Canine leptospirosis is a bacterial disease caused by spirochetes of the genus Leptospira. Infections can vary from asymptomatic and chronic infections to clinical acute diseases. The disease is endemic in tropical areas, such as Latin American countries, but a broad understanding of the dynamics of circulation of strains, based on molecular data, has not yet been performed. Based on in silico analyses, the present study aims to analyze the genetic diversity and circulation patterns of haplotypes from pathogenic leptospires infecting dogs in Latin America. DNA sequences were obtained from GenBank platform, curated, and aligned. Genetic distances were calculated, and a maximum likelihood tree and haplotype network were constructed. According to the inclusion criteria adopted, a total of 148 sequences were identified. Most of the records were from Brazil, including sequences from L. interrogans serogroup Icterohaemorrhagiae. Phylogenetic analysis showed a genetically closely related cluster, consisting of a larger haplogroup that includes the reference strain Fiocruz L1-130, known to be the major circulating strain in humans. Moreover, no genetic variations were observed according to clinical history and/or geographical localization. We described the molecular epidemiology of leptospires circulating among dogs in Latin America and demonstrated a very genetically homogeneous group, elucidating its ubiquitous circulation pattern and drawing attention to the important role of dogs in the One Health transmission dynamics of leptospirosis.

Draft-genome sequences of Leptospira santarosai strains isolated from urogenital tract of cows.

Leptospira santarosai serovar Guaricura is adapted to bovines and is associated with a chronic disease that causes reproductive disorders, leading to important economic losses. Here, we present the draft genomes of three L. santarosai strains isolated from vaginal fluid and one from the urine of cows with reproductive disorders from Brazil.

Successful treatment with streptomycin of genital leptospirosis in naturally infected cows under field conditions.

The single-dose protocol of streptomycin treatment has been recommended to treat renal leptospirosis in bovines. However, treating genital infection remains a challenge. Recently, a protocol using three doses of streptomycin demonstrated effectiveness in the genital clearance of experimentally infected ewes. Therefore, the present study aimed to apply this three-dose protocol for genital infection treatment in naturally infected cows under field conditions. Thirty beef cows were diagnosed as positive by lipL32-PCR in their genital samples. Nucleotide sequences (n = 10) characterized them as Leptospira interrogans sg Sejroe, genetically related to Hardjoprajitno strains. After molecular diagnosis, 13 cows received a single dose of 25 mg/kg streptomycin. The other 17 cows were submitted to the three-dose protocol. The successful treatment rate of genital infection on the single streptomycin dose was 7/13 (53.8%), while the cows that received the three doses 16/17 were negative (94.1% of efficacy). Based on those results, we conclude that the standard treatment preconized for renal infection is not adequate for genital infection, and the three-dose protocol was successful in eliminating the carrier status of genital leptospirosis.

Field application of a combined serological-molecular testing protocol for diagnosing genital leptospirosis in naturally infected cows with gestational losses.

BACKGROUND: Bovine genital leptospirosis (BGL) causes chronic reproductive disease in cattle. This study aimed to apply a combined serological-molecular testing protocol under field conditions for diagnosing BGL in cows with gestational losses. METHODS: Three beef herds with reproductive failures were studied, and 60 cows with gestational losses (20 from each herd) were randomly selected for laboratory diagnosis of BGL. In addition, 40 cows with normal pregnancy were included as a control. Blood samples were collected from all 100 cows for microscopic agglutination testing, and cervicovaginal mucus (CVM) samples were collected from 28 cows with gestational losses and 20 control cows for lipL32-PCR. RESULTS: All herds had high Leptospira seroreactivity (>65%), mainly against serogroup Sejroe. Ten of the 28 CVM samples from cows with gestational losses were PCR-positive, while all samples from the control group were negative (p < 0.05). LIMITATIONS: Unfortunately, the positive samples did not amplify in secY-PCR for nucleotide sequencing, which would allow the identification of leptospiral strains. CONCLUSION: Serology was sufficient to indicate leptospirosis at the herd level, but the definitive diagnosis of BGL was only possible using CVM PCR. Although seroreactivity against serogroup Sejroe has been associated with gestational losses, this is the first study to conduct CVM PCR as a confirmatory test for BGL diagnosis in extensive beef herds under field conditions.

Pinnipeds carriers of pathogenic Leptospira: New data based on molecular characterization.

Leptospirosis is a bacterial disease caused by the infection of pathogenic strains of the genus Leptospira, endemic in tropical and subtropical regions. Although well documented in terrestrial animals and humans, little information is available on its distribution and impact on marine animals. Despite clinical manifestations that may occur, the occurrence of carriers was suggested in some species. Nevertheless, there are few studies regarding the infection by Leptospira sp. in marine mammals. In this context, and considering the One Health approach, the present aimed to investigate pinnipeds' role as Leptospira sp. carriers. Kidneys of 47 pinnipeds of two species, Arctocephalus australis (n = 40) and Arctocephalus tropicalis (n = 7) were collected. DNA was extracted and the diagnosis was performed through LipL32-PCR and genetic characterization based on secY gene sequencing. Phylogenetic analysis and haplotype networks were constructed. Pathogenic Leptospira sp. DNA was detected in 31.9% (15/47) of the tested pinnipeds. It was possible to amplify and sequence eight strains (6 for A. australis, 2 for A. tropicalis), all identified as L. interrogans, with high similarity with sequences from Icterohaemorrhagiae serogroup. Phylogenetic analysis revealed sequences from the present study grouped in species-specific unique clusters, but very close to others from humans, wild animals, and domestic animals. We demonstrate that pinnipeds could act as carriers, and play an important role in leptospirosis dynamics.

Unconventional Sites for Diagnosis of Leptospirosis in Bovine Anicteric Fetuses.

BACKGROUND: Bovine leptospirosis is an important reproductive disease and abortion is a major sign, leading to economic impacts. Due to its multifactorial etiology, the proper diagnosis of the cause of the abortion is crucial. Necropsy of the fetuses followed by molecular analysis is recommended for diagnosis, and the investigation mainly occurs in the kidneys and liver. This study aimed to analyze unconventional sites for the presence of leptospiral DNA in bovine anicteric aborted fetuses. METHODS: Five fetuses of the same herd were received for necropsy and diagnosis. Conventional lipL32-PCR was performed in the fetuses' kidneys, livers, lungs, hearts, spleens, subcapsular kidney content, abomasal fluid, and in the cavity's hemorrhagic contents. To complete the investigation, the sera of 30 cows of the herd were collected to perform the serologic screening by Microscopic Agglutination Test. In addition, six subfertile non-pregnant cows from the same herd were selected due to their low reproductive performance, and genital samples (uterine fragment and cervicovaginal mucus) and urine were collected for lipL32-PCR. PCR-positive samples were submitted to a nested PCR of the secY gene and intended for sequencing. RESULTS: The herd presented seroreactive animals (11/30, 36.6%), all against the Sejroe serogroup, with titers between 200 and 1600. In necropsy, four fetuses showed hemorrhagic and anicteric lesions, while one fetus had no macroscopic lesions. Regarding molecular analysis, all the fetuses were positive in lipL32-PCR and the positive sites were the heart, lungs, subcapsular kidney content, thymus, kidneys, liver, and abomasal fluid. Only one fetus presented positive results in the kidney and liver, while three fetuses were positive in the abomasal fluid. Five of six cows were positive for lipL32-PCR, all being positive only in genital samples. Of the fetuses and the cows, seven sequences were obtained and all were identified as Leptospira interrogans serogroup Sejroe serovar Hardjoprajitno. CONCLUSIONS: In order to improve the diagnosis of leptospirosis in cows, it is recommended to perform a comprehensive analysis of the samples, beyond the kidneys and liver. Thus, we highly encourage testing multiple organs by PCR to investigate abortions suspected of bovine leptospirosis, particularly in anicteric fetuses.

Equine genital leptospirosis: Evidence of an important silent chronic reproductive syndrome.

Leptospirosis may be associated with important syndromes in equines, including reproductive ones. Recently, our group demonstrated a reproductive syndrome of leptospirosis in ruminants (Bovine Genital Leptospirosis - BGL), that presents its own agent, particular diagnostic methods, and specific treatment and prevention, requiring special attention. We realized that the genital syndrome of leptospirosis may not be exclusive to ruminants and may also affect horses. The aim of the present study is to gather the available data on leptospirosis in equine reproduction and propose a new genital leptospirosis syndrome in horses, which we have named Equine Genital Leptospirosis (EGL). The main agent is most probably serovar Bratislava (serogroup Australis), whose role has been increasingly recognized in the etiology of infection, presenting a worldwide distribution. The most probable relevant transmission route in the EGL context is the sexual transmission. Subfertility, estrus repetition, and, less commonly, late-term abortions are the most common effects. Serology may not be adequate to diagnose this chronic silent reproductive leptospiral infection. As most studies regarding equine leptospirosis investigate urinary samples, EGL is vastly underdiagnosed, and the disease is little studied and underestimated. Therefore, PCR, particularly from cervicovaginal mucus (CVM) and uterine fragments, turns out to be a rapid and definitive diagnosis choice. In order to mitigate the effects of the disease in equines, triad antibiotic therapy, vaccination, and environmental management are essential. Adequate recognition of the particularities of this syndrome may help to highlight its occurrence and contribute to a broader understanding of its clinical manifestations, use of a correct diagnosis, and development of novel approaches for its control.

The Efficacy of Vaccination in the Prevention of Renal and Genital Leptospirosis in Experimentally Infected Sheep.

(1) Background: Leptospirosis, mainly determined by strains belonging to serogroup Sejroe, has a direct impact on the reproductive efficiency of ruminants, such as sheep. In Brazil, Leptospira santarosai serovar Guaricura, known to be able to chronically colonize the uterine environment, is of special note. Although vaccination minimizes the effects of acute disease, whether or not it can protect from renal colonization remains controversial, and its effects on the genital tract are unknown. In this context, the present study aims to investigate the efficacy of vaccination in the prevention of experimental genital leptospirosis. (2) Methods: Eighteen sheep were divided into three groups: one vaccinated with a polyvalent commercial bacterin, one vaccinated with an autologous bacterin, and one unvaccinated. After 14 days, the sheep were experimentally challenged with 108 leptospires (L. santarosai, serogroup Sejroe, serovar Guaricura, strain FV52) intraperitoneally. Serology and colonization of the urinary and genital tracts were carried out monthly by PCR for 210 days in all animals. (3) Results: Vaccination significantly elicited antibody titers with a predominance of agglutinins against serogroup Sejroe. Both urinary and genital infections were confirmed by PCR. Positivity in urine PCR was absent after D120, while genital infection persisted until the end of the study. Serological results and the finding that vaccination did not avoid renal colonization align with previous studies. Despite vaccination, Leptospira established chronic and asymptomatic colonization of the genital tract until D210, an outstanding finding that remains to be fully understood in its mechanisms. (4) Conclusions: This is the first study conducted to analyze the effects of vaccination in the prevention of genital leptospirosis.

Bovine genital leptospirosis: Evidence of ovarian infection by Leptospira interrogans.

Leptospirosis in ruminants causes reproductive failures leading to important economic losses. This study assessed the occurrence and genetically identified Leptospira spp. in the follicular fluid (FF) of naturally infected live cows. A total of 251 asymptomatic cows from different commercial dairy herds were subjected to ovum-pick up technique for follicular fluid sampling. PCR was performed for Leptospira spp. detection and phylogenetic analysis was later implemented for sequencing. From 251 samples analyzed, 67 (26.7 %) were lipL32-PCR positive, confirming the presence of leptospiral DNA on FF. Furthermore, it was possible to amplify and sequence nine strains after secY nested-PCR. All of them were identified as L. interrogans, with 100 % of identity with strains belonging to Sejroe serogroup. Our findings reveal a high occurrence of infection of Leptospira in the ovarium of asymptomatic cows, highlighting the importance of considering the silent leptospirosis syndrome when screening animals for assisted reproductive biotechniques.

Identification of vaginal Leptospira in cervical-vaginal mucus of slaughtered pigs in the amazon region.

Swine genital leptospirosis is an infectious disease that leads to economic losses due to abortions, stillbirths, and reproductive failures. Considering the scarcity of studies regarding this condition, the objective of the present study was to identify and analyse leptospires infecting the reproductive tract of female pigs slaughtered in the Amazon region. Cervical-vaginal mucus (CVM) from 150 non-pregnant females were collected and submitted to molecular analysis. Initially, polymerase chain reaction (PCR) based on the lipL32 gene was performed. A total of 26.7% (40/150) samples were positive, indicating the presence of Leptospira sp. DNA. Subsequently, positive lipL32-PCR samples were evaluated using secY nested-PCR and sequencing procedures. Eleven amplicons could be sequenced and were identified as Leptospira interrogans (100% identity). Results from phylogenetic analyses led to identification of a putative strain of L. interrogans serogroup Australis, which is indicative of this being a serogroup. In the present study, there was detection of female pigs with leptospires in CVM indicating the possibility of venereal transmission. The large number of genital positive cases could indicate that genital leptospirosis syndrome could also be relevant onto swine production.

Leptospira noguchii associated to reproductive disease in ruminants.

Leptospirosis is known to determine reproductive disorders on livestock, and Leptospira interrogans and Leptospira borgpetersenii are the most frequently reported species. Leptospira noguchii is an emerging pathogen, but its association with reproductive disease is unclear. We have detected L. noguchii as the agent of an outbreak with reproductive disorders in a Brazilian dairy goat flock. In the kidding season, five out of 10 Saanen had abortions in the final month of pregnancy and two newborn kids had acute clinical signs. After necropsy of three foetuses and one newborn kid, fragments of liver, lung and kidney were submitted to lipL32-PCR. It yielded positive results in at least one fragment from each animal. After, a nested secY-PCR, followed by sequencing, could identify L. noguchii, with 99-100% of identity with sequences obtained from cattle in the same region. For the first time, L. noguchii was detected in goats and, most importantly, the association of this leptospiral species with reproductive failures in ruminants has been demonstrated.

Characterization of leptospiral DNA in the follicular fluid of non-pregnant cows.

BACKGROUND: Bovine leptospirosis is mainly characterized by reproductive disorders. Contamination of the oocyte was previously demonstrated in vitro, resulting in some apparent damage. However, it is not clear whether it occurs under natural conditions. The present study aimed to characterize the presence of pathogenic Leptospira DNA in the ovarian follicles of non-pregnant cows. METHODS: Follicular fluid samples were collected from 65 animals and subjected to lipL32 PCR and secY sequencing. RESULTS: In total, seven of 65 (10.8%) were positive, indicating a possible early infection of the oocyte. Moreover, secY sequencing identified L. interrogans and L. santarosai, both very closely related to bovine strains from the Sejroe serogroup (Hardjoprjitno and Guaricura). We demonstrated that ovarian follicles can also be infected. CONCLUSIONS: It was hypothesised that ovarian infection can contribute to embryonic death, causing reproductive failure and estrus repetition. In the present study, we show that the organism identified in the follicle is closely related to one that is known to be associated with reproductive disorders.

Bovine Genital Leptospirosis and reproductive disorders of live subfertile cows under field conditions.

Bovine genital leptospirosis (BGL) is characterized by silent chronic reproductive disorders, most related to early embryonic death leading to estrus repetition, subfertility and abortions. However, most studies were conducted in slaughterhouses, which lacks reproductive and sanitary history of the studied animals. This study aimed to evaluate the occurrence of Leptospira sp. infection in live cows with history of low reproductive efficiency. Blood, urine, cervico-vaginal mucus and uterine fragment were collected from nine cows of the same herd presenting reproductive failure (abortions, estrus repetition and chronic infertility). Serology (MAT) and molecular analysis (PCR and nucleotide sequencing) were performed. Serology showed three (33.3%) seroreactive cows, two to Sejroe and one to Icterohaemorrhagiae serogroups. Six cows (66.7%) presented leptospiral DNA on genital samples, while all urine samples were negative. L. interrogans was identified in five samples, very closely related to strains from Sejroe (n = 3) and Icterohaemorrhagiae (n = 2) serogroups, while L. noguchii was identified in one sample. Results from this preliminary study demonstrates the presence of leptospires on uterus and reinforces the negative impact of leptospiral infection on reproductive tract, highlighting its association with reproductive failures on live animals.

Extra-renal bovine leptospirosis: Molecular characterization of the Leptospira interrogans Sejroe serogroup on the uterus of non-pregnant cows.

Bovine genital leptospirosis is a chronic disease that causes reproductive disorders such as abortions, stillbirths, and estrus repetition, as well as economic losses. Despite clinical signs related to reproductive failure, the majority of studies have focused on the detection of Leptospira spp. in the urine, while few have considered the reproductive tract. Consequently, the aim of the present study was to investigate the uterus as an important extra-renal site of leptospiral infection in cows. A total of 42 non-pregnant cows were studied at a slaughterhouse. Blood samples and uterine fragments were collected for serology and molecular analysis, respectively. Concerning serologic results, 20.5 % presented as reactive, all of them against the Sejroe serogroup. Regarding lipL32 PCR, 26.2 % (11/42) of samples were positive for pathogenic Leptospira sp. Sequencing the secY gene short region enabled nine strains to be characterized, all of which were L. interrogans, with high identity (98.8 %-99.8 %) with serovar Hardjo. The use of molecular tools substantially improved the sensitivity of Leptospira sp. detection at species level and demonstrated that the uterus is an important site of bovine leptospiral infection. The findings of the present study reinforce our understanding that leptospiral uterine infection are associated to members of the Sejroe serogroup.

Acanthocephalan parasites of the flounder species Paralichthys isosceles, Paralichthys patagonicus and Xystreurys rasile from Brazil.

Flounders are commercially and economically important fish. A total of 120 specimens of flounders (60 Paralichthys isosceles, 30 Paralichthys patagonicus and 30 Xystreurys rasile) were collected off the coast of the state of Rio de Janeiro, Brazil. The fish were measured, necropsied and filleted, and then had their organs investigated for acanthocephalans. Taxonomic identification of the parasites was based on morphological, morphometric and genetic characters. Paralichthys isosceles and P. patagonicus were parasitized by juveniles of Serrasentis sagittifer, Bolbosoma turbinella, Corynosoma australe and C. cetaceum; Xystreurys rasile was parasitized by C. australe. Genetic characterization confirmed the identification of specimens of Bolbosoma turbinella and Corynosoma australe, as demonstrated by phylogenetic analyses using both ITS and cox1 molecular targets. Parasite indices of prevalence, intensity, mean intensity, abundance, mean abundance, and range of infection, as well as infection site, were evaluated for each parasite species. This is the first report of S. sagittifer parasitizing P. isosceles and P. patagonicus, and B. turbinella parasitizing P. patagonicus.