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.

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.

Serological and molecular characterization of Leptospira kirschneri serogroup Grippotyphosa isolated from bovine in Brazil.

Leptospirosis is a zoonotic disease caused by a bacterium of the genus Leptospira that affects domestic animals, including cattle. Despite of this importance in livestock, little is known about the epidemiology of leptospirosis in cattle in Brazil and other tropical countries. In this context, the objective of this study was to perform the culture of urine and kidney (n = 372 animals) of slaughtered bovines of Minas Gerais state (Brazil), in order to obtain pure cultures of Leptospira strains and carry out its characterization by serological (microscopic agglutination test) and molecular techniques (rrs, secY sequencing and variable-number tandem-repeat genotyping). One strain, named UFU 02, was obtained from a male bovine without apparent alterations in ante-mortem examination. The strain was characterized belonging to species Leptospira kirschneri and serogroup Grippotyphosa. The serogroup Grippotyphosa is most often described in wild species, suggesting a link between domestic and wild cycles/environments. Considering the importance of the country in livestock breeding, the knowledge about leptospiral strains could help to control the disease in the herds.

Atypical virulence of Leptospira kirschneri serogroup Icterohaemorrhagiae isolated from capybaras (Hydrochoerus hydrochaeris) in hamster model.

Leptospirosis is an important zoonotic disease and, in urban areas, rodents are considered the main reservoir of Leptospira to human hosts. It has been described that capybaras, the world largest rodent, also harbor and shed leptospires by urine. Although not virulent to their hosts, strains of rodent origin are virulent for the hamster. In this context we aim to investigate the virulence of Leptospira kirschneri strains of serogroup Icterohaemorrhagiae recovered from capybaras in Brazil in the hamster model. Five isolates of Leptospira recovered from asymptomatic capybaras were submitted to virulence tests following the suggested protocols and the 3Rs policy for experimental science. Briefly, 1 ml of 1 × 108 leptospires was inoculated intraperitoneally four times in one hamster for each strain. Four days after inoculation, a blood sample was collected via the gingival route for confirmation of blood culture infection. The infected animals were kept isolated in microisolators to observe clinical signs and monitored daily till day 21 post-inoculation. None strain caused acute disease in hamsters but were able to colonize their kidneys. The present study demonstrated that although Icterohaemorrhagiae strains are often reported as virulent, not all strains of that serogroup are indeed aggressive. Concluding, we report that strains of L. kirschneri serogroup Icterohaemorrhagiae recovered from healthy capybaras presented an atypical virulence to the hamster model, what reinforces that virulence is an intrinsic strains characteristic.

A systematic review on the distribution of Mycobacterium bovis infection among wildlife in the Americas.

The occurrence of Mycobacterium bovis infection in wildlife places at risk livestock, public health, and ecosystems that house endangered species. However, data on wild species that may act as possible reservoirs in the Americas are scarce. This systematic review analyses the available data on wildlife in the Americas regarding the infection by M. bovis. We searched articles published in indexed journals using the keywords: "Mycobacterium bovis," "wild," and "animals". After applying the keywords using online databases, during March and August of 2018, we found 12 articles which encompassed 15 species of wild animals, of which three consisted of wild ruminants. The evidence showed that M. bovis is present among the wild animals in the Americas. The methodological limitations for diagnosing M. bovis in wild animals are many, demanding the development of new and more precise tools. Furthermore, new researches are needed to elucidate the role of the wild animals in the epidemiology of M. bovis and its possible impact on production animals and public health.

Occurrence of uterine carriers for Leptospira interrogans on slaughtered cows.

Reproductive tract is an important site of infection for chronic leptospirosis and cooperate in pathogenesis of reproductive failure, leading to economic losses. Since serology techniques cannot detect chronic carriers, the molecular analysis of clinical samples is an alternative to detect these animals on livestock. The aim of the present study was to perform a retrospective study in order to detect leptospiral uterine carriers in slaughtered cows. Tissue samples were collected from 50 post-pubertal, nonpregnant cows. These samples were fixed in 10% buffered formalin, paraffin-embedded and stored. PCR targeting lipL32 gene and molecular characterization by secY sequence was performed. Leptospiral DNA was identified in 18% (9/50) examined blocks. Two sequences were characterized as L. interrogans. These findings suggest that the presence of infectious leptospires in uterus is associated with the physiopathogenesis of the reproductive failure.

Susceptibility among breeds of sheep experimentally infected with Leptospira interrogans Pomona serogroup.

Leptospirosis is a disease that negatively affects the productive and reproductive indices of ruminants. Sheep are considered highly resistant to infection, although susceptibility may vary among breeds. Thus, the aim of the present study was to analyze the susceptibility between sheep breeds to the experimental infection by leptospires of the Pomona serogroup. Pomona serogroup, Kennewicki serovar strain (1 × 107 bacteria) was inoculated via the conjunctival route in 12 sheep divided into two groups, one comprising Santa Inês ewes and the other comprising crossbred sheep. In each group, five ewes were challenged with the bacterial strain and one was used as control. All sheep were monitored for 60 days, during which blood samples were collected for serological diagnosis and urine and vaginal fluid samples for molecular and microbiological analyses. Finally, as ewes were submitted to euthanasia and necropsy, some tissues of interest were collected for microbiological, molecular, and histopathological diagnoses. The groups were compared regarding the number of positive reactions according to diagnostic tests. All sheep in each group presented antibodies to Leptospira in all serological analyses, except animals of the control group. However the Santa Inês sheep presented higher concentration and duration of the titers, and their positive reactions were detected earlier than those in crossbred sheep. The antibody titers in group A (median 200, geometric mean 317.48) were significantly different from the group B (median 800, geometric mean 918.96) at D60 post-infection (P = 0,032). The Santa Inês sheep presented a higher number of positive reactions than did the crossbred sheep in the molecular diagnostic tests. According to the molecular diagnosis, the Santa Inês sheep presented more reactions (urine and vaginal fluid) compared to crossbred ewes, but there was no predominance in the detection of leptospiral DNA when comparing urine and vaginal fluid results, nor even between the number of positive kidneys and uterus. The Santa Inês sheep presented a higher number of positive bacteriological cultures. No sheep in either group presented alterations in anatomopathological and histopathological findings. Pure-bred sheep may be more susceptible than crossbred sheep to infection by Leptospira sp. Our findings emphasize the importance of the genital tract as a site of extraurinary infection and indicate the possibility of venereal transmission in the species.

Diagnosis of acute canine leptospirosis using multiple laboratory tests and characterization of the isolated strains.

BACKGROUND: Dogs presenting with acute leptospirosis may present non-specific clinical and laboratory findings, and the definitive diagnosis may require additional confirmatory tests, including bacterial culture, for the direct or indirect identification of the pathogen. The present study describes the diagnosis of leptospirosis in suspected dogs based on the use of multiple diagnostic tests, including serological, molecular and bacteriological tests, along with the characterization of the recovered leptospiral strains. RESULTS: Urine, serum and blood samples were collected from 33 dogs with suspected clinical leptospirosis treated at the University of São Paulo Veterinary Hospital Service (Hovet FMVZ-USP) between 2013 and 2016. Only dogs with high blood urea nitrogen and creatinine levels in association with multiple clinical manifestations of the disease were included. Leptospiral culture, PCR and serology (Microscopic agglutination test - MAT) were performed in blood and urine samples taken from all suspected dogs at clinical presentation, and an additional prospective MAT titration was performed in seven dogs. Infection could be identified exclusively by PCR in 10 dogs (30.3%), exclusively by MAT in four dogs (12.1%) and by both tests in four dogs, totaling 18 dogs (54.5-95%CI: 37.6-71.5). Six out of eight MAT-confirmed cases presented with the highest titers against the Icterohaemorrhagiae serogroup. Leptospires were recovered from urine samples from two PCR-positive dogs, and both strains could be characterized by Multilocus Sequence Analysis and serogrouping as L. interrogans serogroup Icterohaemorrhagiae. Both isolates were shown to be pathogenic in the hamster model. CONCLUSIONS: The simultaneous use of MAT and PCR was able to increase the diagnosis of leptospirosis in clinically suspected cases. Despite the increasing incidence of new serovars affecting dogs being reported in different locations, our results suggest that leptospiral strains belonging to the Icterohaemorrhagiae serogroup are still a major causative agent of canine leptospirosis in São Paulo, Brazil.

Genomic characterisation of Leptospira inadai serogroup Lyme isolated from captured rat in Brazil and comparative analysis with human reference strain.

Leptospira inadai is classified as a species of the Leptospira intermediate group that has been poorly studied due to its apparent insignificance to human and animal health. Nevertheless, over the last two decades the species has been described in human cases in India and in carrier animals in Ecuador. Here, we present the first identification and genomic characterisation of L. inadai serogroup Lyme isolated from captured rodent in Brazil. Even though the M34/99 strain was not pathogenic for hamsters, it was able to establish renal colonisation. The M34/99 strain presented high similarity with L. inadai serogroup Lyme human reference indicating that animal strain could also infect humans, although it does not represent high risk of severe disease. An extrachromosomal sequence was also identified in M34/99 strain and presented high identity with previously described L. inadai phage LinZ_10, suggesting that phage-like extrachromosomal sequence may be another feature of this understudied species.

Comparative genomics of pathogenic Leptospira interrogans serovar Canicola isolated from swine and human in Brazil.

Leptospira interrogans serovar Canicola is one of the most important pathogenic serovars for the maintenance of urban leptospirosis. Even though it is considered highly adapted to dogs, serovar Canicola infection has already been described in other animals and even a few human cases. Here, we present the genomic characterisation of two Brazilian L. interrogans serovar Canicola strains isolated from slaughtered sows (L0-3 and L0-4) and their comparison with human strain Fiocruz LV133. It was observed that the porcine serovar Canicola strains present the genetic machinery to cause human infection and, therefore, represent a higher risk to public health. Both human and porcine serovar Canicola isolates also presented sequences with high identity to the Chinese serovar Canicola published plasmids pGui1 and pGui2. The plasmids identification in the Brazilian and Chinese serovar Canicola strains suggest that extra-chromosomal elements are one more feature of this serovar that was previously unnoticed.

Detection of bovine carriers of Leptospira by serological, bacteriological, and molecular tools.

Bovine leptospirosis is an important infectious disease that causes reproductive problems and economic risks, particularly in the tropics. The present study aimed to determine the extent of Leptospira infection among bovines on a slaughterhouse from Rio de Janeiro, Brazil via serological, bacteriological, and molecular tests. Two hundred eight bovines were examined in total, and we obtained 208 blood samples for serology, 198 urine samples collected via direct bladder puncture for polymerase chain reaction (PCR) and culture, 208 kidney samples (one from each animal) for PCR and culture, and 92 vaginal fluid samples from sterile swabs for PCR and culture. Serology demonstrated that 77/208 (37%) of the animals presented anti-Leptospira antibodies. Serogroup Sejroe was by far the most common. One hundrd thirty-three animals (63.9%) were PCR positive in at least one of the tested samples and were considered as Leptospira carriers. Furthermore, ten isolates were obtained by pure culture, all of them from urine samples. Bovine leptospirosis is widely prevalent, and the occurrence of renal carriers was unexpectedly much higher than generally reported.

A systematic review of leptospirosis on wild animals in Latin America.

Leptospirosis is a bacterial systemic infection which affects domestic animals and wildlife, as well as humans. Many wild animals act as reservoirs of leptospires. Nevertheless, the real role of wildlife animals as source of infection to livestock and humans, as well as the most important reservoirs and leptospiral strains remains unclear. This systematic review assesses the available data about wildlife and their biomes in Latin America, concerning to leptospiral infection. In addition, we discuss the development of the research on leptospirosis in wildlife in this region. After the application of exclusion criteria, 79 papers were analyzed, comprising 186 species, 122 genus, 53 families, and 19 orders from four classes. Mammals were the most studied class, followed by Amphibian, Reptile, and Aves. The Icterohaemorrhagiae serogroup was predominant in most biomes and many orders. A small number of antigens detected the majority of seroreactive animals of each class, and a smaller panel may be used at microscopic agglutination test. Further studies must always consider edaphoclimatic conditions besides only host class or species, in order to obtain a broader understanding of the wild epidemiological cycle of leptospirosis in the region. In conclusion, direct and indirect evidences demonstrate that leptospirosis is largely widespread among wildlife in all biomes of Latin America. Moreover, more research on the role of wildlife on the epidemiology of leptospirosis and its impact on livestock and public health are required, particularly focusing on direct detection of the agent.

Detection of anti-Leptospira inhibitory antibodies in horses after vaccination.

Leptospirosis is a relevant zoonosis that affects the reproductive performance of livestock, impairing the economy. Few studies have demonstrated the effects of vaccination against leptospirosis on naturally exposed horses. This study aimed to detect anti-Leptospira inhibitory antibodies in horses after vaccination. A total of 54 mares were studied using Growth Inhibition Test (GIT) in three moments. The present results demonstrate the usefulness of GIT for confirming inhibitory effects of specific antibody production. Results have also demonstrated that vaccination positively influenced on the presence of inhibitory antibodies in horses.

Seronegativity of bovines face to their own recovered leptospiral isolates.

Leptospirosis is an important cause of reproductive failure in cattle. The standard diagnostic tool (MAT) is recommended for herd but not for individual diagnosis. The aim of this study was to evaluate the humoral response of bovines face to their own recovered isolates. A total of 25 bovine from which leptospires were recovered were tested by MAT against reference strains and their own isolates. Only three cows (12%) presented seroreactivity against their own isolates. This study demonstrates that cattle may not react against their own isolates and highlights the importance of interpreting serological negative results with caution.

Bovine leptospirosis: Prevalence, associated risk factors for infection and their cause-effect relation.

Leptospirosis is a cosmopolitan infectious disease that causes severe reproductive disorders in cattle, especially those related to abortion. This disease has rodents as main reservoirs; however, cattle are responsible for maintenance of the disease. Thus, the aim of this study was to identify the factors associated with infection and cause-effect relation of leptospirosis in dairy herds from Southern of Brazil. Serum samples of 1242 cows were collected from herds classified as of medium and high density, and tested by microscopic agglutination test (MAT). These farms were located in the West part of Santa Catarina State (Brazil). A total of 80 cows (6.44%) were considered positives for the infection with titration of 1:100. Using a multivariate analysis, we identified two factors associated to bovine leptospirosis: dog access to pastures (p < 0.001) and feed exposure to rodents (p = 0.05). Cause-effect analysis demonstrated that the occurrence of reproductive disorders was significantly (p = 0.01) linked to leptospirosis. Thus, we conclude that leptospirosis is prevalent in dairy cattle in the west part of Santa Catarina state, as well as the access of dogs to pastures and contact of rodents with feed increase the chance of cattle infection by Leptospira spp.

Occurrence of oxidative stress in dairy cows seropositives for Brucella abortus.

Bovine brucellosis is an important zoonotic disease caused by the bacterium Brucella abortus that leads to economic losses due to animal discard and commercial restrictions. Since positive animals for brucellosis are culled, little is known about the pathogenesis of this disease. Therefore, the aims of this study were to evaluate possible changes in the activity of deaminase adenosine (ADA) and the oxidative stress in cows seropositives for brucellosis (Experiment I), and to evaluate the seroprevalence of B. abortus in dairy cows from the Western state of Santa Catarina, Southern Brazil (Experiment II). The Experiment I evaluated 20 pregnant cows: ten seropositives for B. abortus and ten seronegatives that were used as controls. The ADA activity and markers of oxidative stress (TBARS, catalase (CAT) and superoxide dismutase (SOD)) were evaluated in these animals. A reduction in the activity of ADA and catalase enzymes in seropositive animals was observed (p < 0.001). Conversely, there was an increase in TBARS levels and superoxide dismutase activity in cows infected by B. abortus (p < 0.001). The presence of oxidative stress and a reduction of ADA might be related to the modulation of the inflammatory response. The experiment II was performed due to a high number of herds with restrictions imposed by cases of brucellosis in the state of Santa Catarina in the last two years, and thus, the seroprevalence for B. abortus was evaluated in 1242 serum samples of cows of 69 herds. The serodiagnosis was performed using two tests: buffered acidified antigen and 2-mercaptoethanol. However, none of the serum samples were positive for B. abortus. Although we did not find seropositive animals for brucellosis in our study, the disease still requires continued surveillance, due to its economic impact, and to the oxidative stress caused by it, which may have contributed to cases of abortion in three seropositive cows (Experiment I) in the final third of the gestation.

Antibiotic resistance in Staphylococcus sp. isolated from the vaginal environment of squirrel monkeys (Saimiri spp.) bred ex situ.

BACKGROUND: Squirrel monkeys (Saimiri spp.) have been widely used as animal models; however, the occurrence of Staphylococcus sp in their vaginal microbiota remains to be described. METHODS: Samples were collected from 175 adult squirrel monkeys to isolate Staphylococcus sp and to test for susceptibility to a panel of nine antimicrobial agents. RESULTS: Isolates with characteristics of the genus Staphylococcus were detected in 95 of 175 samples. Coagulase-negative staphylococci (CoNS) were the most common (95.8%, 91/95) isolates. Resistance to antibiotics was observed in 47.3% (45/95) of isolates. Resistance to tetracycline was observed in 28.5% (26/91), chloramphenicol in 15.4% (14/91), and methicillin in 13.2% (12/91) of CoNS. Coagulase-positive staphylococci were resistant to tetracycline, erythromycin, and methicillin. CONCLUSIONS: The presence of Staphylococcus sp in vaginal samples obtained from squirrel monkeys suggests that these animals were in a carrier state. Furthermore, isolating strains resistant to methicillin reinforces the biosafety care of a colony.

A systematic review of leptospirosis on dogs, pigs, and horses in Latin America.

Leptospirosis is a worldwide zoonosis which can affect many species. Control programs need accurate diagnosis to be successful, and currently, diagnosis relies on serology. It presents three main issues: the sampling, the antigen panel, and the cutoff point. Herein, we propose a systematic review on leptospirosis among dogs, pigs, and horses in Latin America in order to improve the understanding of the seroepidemiology of leptospirosis in these species in the region as well as the temporal development of the research on this topic and, consequently, improve the chances of success on control programs. Internet databases were consulted over 2015. Inclusion criteria included serosurvey using MAT; a relevant number of animals; the presence in the antigen panel of at least one representative of serogroups Icterohaemorrhagiae and Canicola for dogs, Icterohaemorrhagiae, Australis, and Pomona for pigs, and Icterohaemorrhagiae and Australis for horses; and a cutoff point of ≥100. Overall, 240 papers were studied, of which 87 referred to dogs, 66 to pigs, 39 to horses, and 48 to more than one of the studied species. In relation to those that met all the inclusion criteria, it was 45 (66.2%) in dogs, 23 (41.8%) in pigs, and 23 (63.9%) in horses. Leptospirosis is widespread in Latin America. Predominant serogroups are Canicola to dogs and Icterohaemorrhagiae to pigs and horses. Therefore, research on animal leptospirosis should be encouraged in Latin America, in order to reach a greater standardization in studies and then achieve better results on control programs.