Bovine Genital Leptospirosis: An Update of This Important Reproductive Disease.

Bovine leptospirosis is an important disease that affects the reproductive sphere. Due to its high relevance for the bovine production chain in a worldwide scenario, a better understanding of the disease is crucial to reduce its negative impacts. The main agents are strains from the Sejroe serogroup, such as Hardjo and Guaricura, which lead to renal and genital infection. The genital colonization causes a chronic, silent, and subclinical reproductive syndrome, called Bovine Genital Leptospirosis (BGL). Embryonic death, estrus repetition, subfertility, and abortions are the main signs of BGL condition in females. However, although leptospires have been identified in semen, the manifestation of BGL in bulls remains to be clarified. The recommended diagnosis of BGL includes a serologic screening of the herds using the microscopic agglutination test followed by PCR of genital samples (cervicovaginal mucus, uterine fragment, or semen), especially from animals with reproductive failures. After the identification of carriers, control is carried out considering three steps: antimicrobial treatment of the carriers, environmental and reproductive management, and herd vaccination. Systematic testing, quarantine of newly arrived animals, and usage of antimicrobials in semen diluents or embryo culture media are other sanitary approaches that are encouraged to improve the control of the syndrome. Herein we discuss protocols for an efficient diagnosis and preventive procedures of BGL, which are fundamental to reducing the negative impact of the disease on cattle reproduction and its consequent economic hazards.

Cellular inflammatory response in the bovine uterus by Leptospira infection may be related to embryo death and subfertility.

Bovine leptospirosis is a chronic disease that causes various reproductive disorders and consequent economic losses worldwide, particularly embryo death. Although Leptospira spp. has already been detected in the genital tract of cows, little is known about the uterine cellular immune response or the intrinsic factors that could contribute to that reproductive failure. In this context, the aim of this study was to assess the uterine cellular inflammatory response after the quantification of cytokine IL-6 in bovine uteri naturally infected with leptospires compared to uninfected. Our results demonstrated that uterine tissues infected with leptospires have higher levels of IL-6 compared to uninfected tissues (p < 0.001). It suggests that the presence of leptospires in the bovine uterus can induce a cellular inflammatory response, which may be related to embryo death and consequent subfertility.

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.

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.

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.

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.

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.

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.

Standardization of chronic uterine infection of hamsters by Leptospira santarosai serovar Guaricura, from serogroup Sejroe.

Leptospirosis is an important zoonosis that in cattle is characterized as a reproductive disease. It is well reported that the main agent of bovine leptospirosis worldwide is Sejroe serogroup serovar Hardjo. Reproductive disease in cattle has several gaps in its knowledge and studies with Golden Syrian hamsters, experimentally infected, are limited. Therefore, a protocol that could reproduce the chronic genital disease in hamsters would be extremely valuable for the advance of the knowledge of that syndrome. The aim of this study was to establish an experimental protocol for chronic non-lethal genital infection of female hamsters by L. santarosai serovar Guaricura (Sejroe serogroup), strain 2013_VF52. For this, two concentrations (1.0 × 108 leptospires/mL and 1.0 × 104 leptospires/mL) were used intraperitoneally in female hamsters of 06-08 weeks of age. Hamsters that survived for up to forty days after inoculation were euthanized. Uterine and renal tissues were collected to evaluate leptospires' presence by PCR and culture. The protocol demonstrated that 1.0 × 104 leptospires/mL of the strain determined chronic genital leptospirosis in the hamster model. The standardization of a protocol for chronic genital leptospirosis in hamsters can be extremely useful for the understanding of the physiopathology of the infection, as the distribution of leptospires in the uterus and the agent-host interactions.

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.

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.

Streptomycin treatment of genital carriers of Leptospira in experimentally infected sheep on different estrous phases.

Leptospirosis in ruminants presents as a chronic disease that causes several reproductive disorders leading to severe economic losses. The current recommended treatment can be efficient to eliminate the renal carrier state, however little is known about the effect of this drug in removing the genital carrier state and the hormonal influence on it. A total of 12 primiparous sheep experimentally infected with a strain of Leptospira santarosai serogroup Sejroe, FV52 strain, were used and distributed as group A (estrus; n = 5), group B (metaestrus; n = 4) and group C (control; n = 3). At D0, groups A and B were treated with streptomycin (25 mg/kg) single dose. Samples of cervicovaginal mucus (CVM) were collected on days 0, 3, and 35 post-treatment, while uterine fragment (UF) samples were collected on days 3 and 35, for PCR. Even after antibiotic treatment, all groups presented infected animals, at D3 and D35, with no significant difference between the treated and control groups. Based on these results, it was conducted a second protocol of treatment with streptomycin, IM (25 mg/kg) for three consecutive days, which was 100% effective to eliminate the genital carrier state; therefore, that protocol should be recommended.

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.

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.

Effect of Vaccination against Leptospira on Shelter Asymptomatic Dogs Following a Long-Term Study.

(1) Background: Vaccination of dogs against leptospirosis is of paramount importance, as they ideally must provide not only long-term protection, but also against the renal carrier state of leptospires. This study assessed the post-vaccine humoral response against Leptospira in naturally exposed dogs and effects on renal carrier status. (2) Methods: A total of 118 dogs were studied for 365 days, separated into Group A (vaccinated, n = 94) and Group B (non-vaccinated, n = 24). Group A was subdivided into three groups: A1 with 32 dogs immunized with the vaccine #1; A2 by 32 dogs with #2; and A3 30 dogs with #3. Serology (MAT and IgG-ELISA) and urinary PCR were conducted. (3) Results: Seroreactivity increased at D15 post-vaccination and, regardless of vaccine brand, remained high up to D180, with antibody switch to IgG after D30. A total of 46.8% of animals from Group A were PCR-positive at least once, in contrast to 75% in Group B, regardless of vaccine brand (p < 0.05; OR: 0.3). (4) Conclusions: All commercial vaccines succeeded at eliciting a long-term IgG-based response and were partially effective at protecting against kidney infection.

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.

The role of Leptospira santarosai serovar Guaricura as agent of Bovine Genital Leptospirosis.

Bovine Genital Leptospirosis (BGL) is an important reproductive disease. The main agents are Sejroe strains, particularly the Hardjo genotypes from Leptospira interrogans and L. borgpetersenii. Although other Sejroe strain, L. santarosai genotype Guaricura, has been frequently isolated from asymptomatic and slaughtered cattle, even from vaginal fluid samples, the role of this strain as real agent of BGL remains uncertain. This study aimed to reinforce L. santarosai strain Guaricura as an important BGL agent, through genetic characterization of a uterine isolate from a live subfertile cow. Urine, cervicovaginal mucus (CVM) and uterine fragment (UF) were collected. In a set up field laboratory, urine, CVM and UF were immediately seeded in T80/40LH medium with antimicrobial cocktail STAFF. Cultures were subcultured in T80/40LH without cocktails, stored at 29ºC and weekly examined. DNA from urine, CVM and UF samples were submitted to PCR targeting lipL32 and secY genes. One leptospiral isolate was recovered from uterine sample; it was serogrouped as Sejroe (titre 25,600) and secY sequencing revealed high genetic similarity with L. santarosai strains from Guaricura serovar. The isolation of this strain from uterus of a live subfertile cow represents substantial evidence that L. santarosai strain Guaricura indeed plays an important role as a BGL agent.

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.