Genital route in experimental infection, a promising approach to study genital leptospirosis in ruminants.

Infection by Leptospira sp., mainly strains from the Sejroe serogroup, impairs the reproductive efficiency of ruminants leading to economic losses. Although the majority of experimental studies use the intraperitoneal route of leptospiral infection, it has been suggested that natural infection occurs frequently by sexual transmission. Thus, we assessed the genital route of infection to study genital leptospirosis in the sheep model. A strain of L. borgpetersenii serogroup Sejroe, serovar Hardjobovis was inoculated in 18 ewes, divided into three groups for inoculation: intraperitoneal (n=6; Gip), cervical superficial (genital) (n=6; Ggen) and conjunctival (n=6; Gconj). Monthly, for 90 days, blood samples were collected for serology (MAT) and PCR was performed on urine, cervical-vaginal mucus, and uterine fragments. All ewes were successfully infected, independently of the infection route. Gip and Ggen did not differ throughout the experiment, either on seroconversion or on PCR positivity on urine or genital samples. In contrast, Gconj presented fewer seroreactive animals (P<0.05) and fewer PCR-pos on genital samples than the other groups. The results obtained demonstrated that, although all groups presented both urinary and genital infections, the genital route was more efficient and did not differ from the traditional intraperitoneal. It indicates that genital via, besides being a naturally occurring transmission via, represents a promising and interesting route regarding future studies related to genital leptospirosis in ruminants, and its use should be encouraged.

Placental abnormalities associated with Leptospira interrogans infection in naturally infected mares.

The reproductive features of equine leptospirosis are often neglected. Equine genital leptospirosis is characterized as a silent chronic syndrome, and besides abortions, leads to placental abnormalities, stillbirths, and birth of weak foals. This study aimed to study the occurrence of placental abnormalities associated with Leptospira interrogans infection in naturally infected mares under field conditions. The studied herd had a high occurrence of placentitis and abortions. Ten pregnant mares, eight with placental abnormalities on ultrasonography and were selected. Serum and cervicovaginal mucus (CVM) samples were collected for serology and PCR, respectively. Positive samples in lipL32-PCR were submitted to the sequencing of the secY gene. In lipL32-PCR of CVM, five out of 10 (50%) mares were positive and all were characterized as Leptospira interrogans. Our results highlight the presence of placental abnormalities in the reproductive subclinical leptospirosis syndrome. We encourage field veterinarians to include leptospirosis testing in their reproductive management.

Growth dynamic of Leptospira spp. from Sejroe serogroup in different media formulae.

The culturing of Leptospira strains from bovine clinical samples is challenging and has resulted in some gaps in securing an epidemiological understanding. Strains related to chronic reproductive leptospirosis in cattle belong to the Sejroe serogroup - not only Hardjoprajitno and Hardjobovis but also Guaricura genotypes. This study analyses the growth of Leptospira strains from serogroup Sejroe in different culture media, with the aim of suggesting better culturing approaches. To meet this objective, two culture media were applied: EMJH and T80/40/LH. In addition, three different cocktails of selective agents were chosen. The combinations of medium and selective additives resulted in 10 different tested formulae. The poor performance of Hardjobovis in EMJH indicated that its growth may represent a possible bias when culturing these strains from bovine samples. The most efficient medium for culturing Hardjobovis was T80/40/LH, while T80/40/LH medium + STAFF combination proved to be the best choice for growth, being recommended for obtaining a higher number of these strains from bovines.

An overview on the molecular diagnosis of animal leptospirosis.

The most common presentation of animal leptospirosis is the subclinical and silent chronic form, that can lead to important reproductive disorders. The diagnosis of this chronic form remains a challenge. The aim of the present study is to gather and critically analyse the current information about molecular tools applied to animal leptospirosis diagnosis, particularly the silent chronic presentation of the infection. Regarding clinical specimens, samples from urinary tract were the most used (69/102, 67·7%), while few studies (12/102, 11·8%) investigated samples from reproductive tract. Concerning the molecular methods applied, the most used is still the conventional polymerase chain reaction (PCR) (46/102, 45%), followed by real-time PCR (38/102, 37·2%). The lipL32 gene is currently the most common target used for Leptospira detection, with 48% of studies applying this genetic marker. From all the studies, only few (21/102, 20·5%) performed gene sequencing. According to the majority of authors, current evidence suggests that lipL32-PCR is useful for an initial screening for Leptospira DNA detection in animal clinical samples. Posteriorly, if DNA sequencing could be performed on positive lipL32-PCR samples, we encourage the use of secY gene as a genetic marker. The molecular methods appear as the most important tools for the diagnosis of the chronic silent leptospirosis on domestic animals, reinforcing its evident impact not only on animal reproduction but also on a One Health context.

Short communication: Uterine leptospiral infection is strongly associated to strains of serogroup Sejroe on experimentally infected hamsters.

Leptospira infection is influenced by the host species and the bacterial strain involved. Important differences on their tissue distribution are referred, also depending on the host and the studied strain. Considering tissue distribution of leptospires the majority of the studies focus on a single strain, or strains from the same serogroup with different inoculation doses. Nevertheless, none had associated different serogroups with their tissue distribution. Thus, the present study aims to investigate the hypothesis that tissue distribution of Leptospira spp. on experimentally infected hamsters may vary according to the strain serogroup. Females of Golden Syrian hamsters were used for the experimental infection with Leptospira isolates (n = 13) belonging to serogroups Icterohaemorrhagiae (n = 8) and Sejroe (n = 5). PCRs were conducted in renal, hepatic and uterine tissue. Seven out of the thirteen studied hamsters presented acute clinical signs and were euthanized before the 21° day p.i. (strains VF237, VF52, U81, UFF-SG001, UFF-G19, UFF-B15, L1-130), while the others presented a subclinical infection. Regarding to the PCR results it was possible to observe that, all but one strain (UFF-SG001), which was detected on all the studied tissues, presented some differences on their tissue distribution. All strains could be detected on at least one tissue and a significant association was observed for the occurrence of the strains of serogroup Sejroe in uterus (p = 0.044). The results of the present study indicate that strains of serogroup Sejroe are strongly associated to uterine infection on experimentally infected hamsters.

Novel MLST sequence types of pathogenic Leptospira spp.: Opening the black box of animal leptospirosis in Brazil.

In the current context of the emergence of certain infectious diseases and discussion of the One Health concept for many of these, the study of leptospirosis - both in domestic and wild hosts - cannot be neglected. The study of animal leptospirosis has evolved in recent years. It has been demonstrated that the human-animal-environment interface is more important than previously thought. In the present study, 35 strains of five pathogenic Leptospira species were isolated from different animal species in Brazil and characterized by rrs, secY, and Multilocus Sequence Typing (MLST) sequencing. Phylogenetic inferences were performed and the molecular diversity of the populations (intra- and inter-population levels) was evaluated. Among the five studied species, 18 different sequence types (STs) were found (22 new alleles and 11 new STs). eBURST analysis revealed two clonal complexes (CCs) and seven singletons. A high genetic diversity was demonstrated (H = 0.954 ± 0.017), mainly for the L. santarosai population (H = 0.942 ± 0.034, n = 20). The same strain was identified in different host species, as well as strains with zoonotic potential circulating in the country. Although the difficulty of culturing Leptospira strains is well known, the high variability of the strains found in Brazil highlights the importance of animals in maintaining the biological cycle of the bacterium in nature. Moreover, the selection of autochthonous strains for the development of vaccines becomes a challenge.

Asymptomatic leptospiral infection is associated with canine chronic kidney disease.

Canine leptospirosis is characterized by an acute or chronic disease. Some dogs may act as asymptomatic carriers, keeping the agent in the renal tubules and eliminating it in the urine for an extended period. Chronic kidney disease (CKD) is multifactorial and pathophysiology has been widely discussed. The aim of the study was to investigate whether the occurrence of CKD may possibly be associated with asymptomatic leptospiral infection in dogs in endemic regions. Serology and urine PCR were performed in 16 dogs with CKD and 48 healthy dogs from an endemic area. Dogs with CKD were more frequently shedders (75%) than non-CKD animals (20.8%). Therefore, our results demonstrate that asymptomatic leptospiral infection is associated with canine chronic kidney disease and that differential diagnosis is important for dogs from endemic areas presenting CKD. The early detection of shedders, besides the obvious impact on Public health may also help to improve the animal health and avoid the development of CKD.

Reduced susceptibility in leptospiral strains of bovine origin might impair antibiotic therapy.

Leptospirosis is a worldwide zoonotic disease determined by pathogenic spirochetes of the genus Leptospira. The control of bovine leptospirosis involves several measures including antibiotic treatment of carriers. Despite its importance, few studies regarding antimicrobial susceptibility of strains from bovine origin have been conducted. The aim of this study was to determine the in vitro susceptibility of Leptospira strains obtained from cattle in Rio de Janeiro, Brazil, against the main antibiotics used in bovine veterinary practice. A total of 23 Leptospira spp. strains were investigated for minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) using broth macrodilution. At the species level, there were not differences in MIC susceptibility except for tetracycline (P < 0.05). Nevertheless, at the serogroup level, differences in MIC were observed among Sejroe strains, mainly for ceftiofur, doxycycline and in MBC for streptomycin (P < 0.05). One strain presented MBC values above maximum plasmatic concentration described for streptomycin and was classified as presenting reduced susceptibility. Efficacy of antimicrobial therapy on bovine leptospirosis could be compromised due to occurrence of infection by Leptospira strains presenting reduced susceptibility.

VNTR analysis demonstrates new patterns and high genetic diversity of Leptospira sp. of animal origin in Brazil.

Leptospirosis is a worldwide zoonosis with a broad host range, including humans and domestic and wild animals. The taxonomic classification of Leptospira species is complex. More recently, several molecular tools have been employed in the attempt to group the different strains of leptospires. The objective of this study was the genetic characterization of Brazilian Leptospira sp. isolates obtained from wild and domestic animals. The genotyping of the strains was performed by the variable-number tandem repeats (VNTR) technique, using the Leptospira interrogans/Leptospira kirschneri and Leptospira santarosai protocols. A total of 27 novel strains were characterized and 22 novel VNTR patterns were described. This study suggests a high genetic diversity among the strains obtained from different wild and domestic animals, and reinforces the need for a broad approach aligned to the One Health concept for leptospirosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Animal leptospirosis is an important cause of reproductive failure in livestock and economic losses to producing countries. This study describes several novel variable-number tandem repeats patterns and points out the high genetic diversity of Brazilian strains. Understanding the circulation of strains between animals is essential for the control of leptospirosis in livestock.

Leptospirosis is strongly associated to estrus repetition on cattle.

Although prevalent, the exact impact of infectious diseases on reproductive failures remains to be determined. Among them, leptospirosis has commonly been reported as cause of abortions on outbreaks. Nevertheless, the majority of the animals present a chronic, silent form of the disease, which is characterized by low reproductive efficiency and is frequently neglected. In that context, we conducted a study that aims to estimate the impact of chronic leptospirosis on reproductive disorders on cattle. A total of 25 different dairy herds with history of reproductive losses from Rio de Janeiro, Brazil, were selected. From each herd, a questionnaire was applied and sera from 20 cows were randomly tested for leptospirosis (totaling 500 cows). Chi-square was performed to estimate the association of seroreactivity with reproductive disorders. A total of 32% of the herds were positive, all of them against serogroup Sejroe. Estrus repetition was the most important reported reproductive problem and it was strongly associated to seroreactivity against leptospirosis. Besides, specific vaccination against leptospirosis was an important protection factor against that disorder. In conclusion, control programs including, but not limited to, vaccines must be implemented on those herds in order to reduce reproductive losses, particularly estrus repetition.

Identification of Staphylococcus epidermidis with transferrable mupirocin resistance from canine skin.

Resistance to mupirocin was analysed in Staphylococcus spp. isolated from healthy dogs (n=21) and dogs with pyoderma (n=47) or otitis externa (n=52). Isolates were identified to species level by MALDI-TOF and PCR-RFLP of the groEL gene. One isolate of Staphylococcus epidermidis from the skin of a healthy dog, which harboured a plasmid carrying the mupA gene, was resistant to mupirocin.

Seroprevalence of anti-Neospora caninum antibodies in sheep from the rapidly expanding flock of Rio de Janeiro, Brazil.

Neosporosis is primarily a disease of cattle and dogs, but Neospora caninum has been linked to abortion and neonatal mortality in sheep. Since the economic, clinical and epidemiological importance of the infection in sheep remains uncertain, this work investigated the seroprevalence of anti-N. caninum antibodies and associated factors in the rapidly expanding flock of Rio de Janeiro state. Blood samples from 388 sheep of 12 farms were collected and sera tested by a commercial Enzyme-Linked Immunosorbent Assay. Seroprevalence at the animal-level was of 6.2% (24/388) and, at the herd-level, 50% (6/12) of the studied farms had at least one seropositive animal. Multivariate analysis detected that occasional veterinary assistance (P < 0.05) was significantly associated to higher seroprevalence, which is also associated to age (P < 0.001) and gender (P < 0.0001). Farmers' investments should focus on making technical assistance more frequent and future studies should assess the association of veterinary assistance with anti-N. caninum antibodies in sheep flocks.

High number of asymptomatic dogs as leptospiral carriers in an endemic area indicates a serious public health concern.

Asymptomatic dogs can be potential hosts of leptospirosis. However, the extension of this phenomenon in endemic areas has not yet been clearly defined. This study is aimed at evaluating the role of asymptomatic dogs as carriers of Leptospira in an endemic area of Brazil. A total of 131 male dogs without apparent leptospirosis symptoms were included in the study based on clinical and hematologic exams. Serum and urine samples were collected for microscopic agglutination tests (MAT) and polymerase chain reactions (PCR) targeted the LipL32 gene, respectively. Forty-two dogs (32·1%) presented seroreactivity (titres ⩾100). The serogroup Icterohaemorrhagiae was predominant, representing 92·7% of the seropositive samples. Overall, leptospiral DNA was detected on 26 urine samples (19·8%). PCR positivity was more common (28·6%) on seropositive dogs than on seronegative (15·7%) ones. Nevertheless, MAT was not correlated to PCR (P > 0·05). Age was not associated with seroreactivity, but dogs older than 5 years of age had 4·07 more chances (odds ratio) of being carriers (PCR positive) than younger ones. Although the fact of knowing that asymptomatic dogs can act as leptospiral carriers is not new, the extension of this fact is impressive in an endemic region, and its role and impact on public health cannot be neglected.

Control of bovine leptospirosis: Aspects for consideration in a tropical environment.

Due to the complex and dynamic epidemiology of leptospirosis on livestock, control is still controversial and frustrating. In this context, this paper discusses the main challenges and perspectives for the control of bovine leptospirosis, particularly under tropical conditions. In order to reduce the effects of the disease in cattle, it has been proposed that the control should integrate the trinomial antibiotic therapy (mainly streptomycin); vaccination (whole-cell bacterins); and environmental management. This last element should be carefully considered in tropics. Despite the enormous economic impact of the disease, mainly on its chronic and silent reproductive presentation, research on control programs is not proportional. Conversely, the number of studies regarding the new vaccine strategies, such as recombinant antigens has been increasing and should be encouraged.

High frequency of leptospiral vaginal carriers among slaughtered cows.

Bovine leptospirosis is one of the most important reproductive diseases that compromise the productivity of cattle farming. However, the presence of the agent on vaginal environment is still poorly understood in cattle. Considering this context, the present study aimed to detect the presence of pathogenic Leptospira sp. in vaginal fluid (VF) of cows. VF and urine were collected from 254 cows from a slaughterhouse for bacteriological culture and PCR (lipL32 gene). Overall, eleven pure culture (4.3%) of leptospiral isolates were obtained. Leptospiral DNA was detected in 128 (50.4%) of VF samples and 81 (31.0%) of urine samples, while on 75 (29.5%) it was exclusively in VF and 28 (11.3%) only in the urine. Detection of leptospiral DNA and the recovery of viable leptospires from VF of a high number of cows without apparent symptoms highlight the role of vaginal carriers and indicate that venereal transmission (female-to-male) could occur in that species. Moreover, VF should be encouraged as a valuable sample for diagnosis of bovine genital leptospirosis.

Molecular analysis of leptospires from serogroup Sejroe obtained from asymptomatic cattle in Rio de Janeiro - Brazil reveals genetic proximity to serovar Guaricura.

Bovine leptospirosis causes substantial reproductive failure in cattle, mainly due to infections with serovar (sv) Hardjo infection. Notwithstanding, other serovars from the serogroup (sg) Sejroe could also have important roles in bovine leptospirosis. The objective was to investigate genetic diversity of serogroup Sejroe isolates obtained from asymptomatic cattle in the state of Rio de Janeiro, Brazil. Urine and vaginal fluid (VF) were collected from clinically healthy cattle immediately after slaughter. Five isolates were recovered and characterized (serogrouping) as belonging to sg Sejroe. Sequencing of rrs and secY genes further identified them as Leptospira santarosai. Analysis of secY sequences indicated a high level of sequence homology to sv Guaricura strains. Based on culture and sequence data, we inferred that other members of sg Sejroe may be important in bovine leptospiral infection, particularly genotypes of L. santarosai serovar Guaricura.

Molecular Characterization and Serology of Leptospira kirschneri (Serogroup Grippotyphosa) Isolated from Urine of a Mare Post-Abortion in Brazil.

A strain of Leptospira kirschneri (serogroup Grippotyphosa) was cultured from urine of a mare post-abortion in Brazil and characterized by serogrouping, multiple-locus variable-number tandem repeat analysis, PGFE, and sequencing of genes rrs and secY. Strains of L. kirschneri have apparently never been recovered from horses in tropical area, only in Europe and USA. Knowledge of local epidemiology is important to interpret genetic profiles of leptospires circulating in an area.

Genotyping of Leptospira directly in urine samples of cattle demonstrates a diversity of species and strains in Brazil.

The aim of this study was to identify Leptospira in urine samples of cattle by direct sequencing of the secY gene. The validity of this approach was assessed using ten Leptospira strains obtained from cattle in Brazil and 77 DNA samples previously extracted from cattle urine, that were positive by PCR for the genus-specific lipL32 gene of Leptospira. Direct sequencing identified 24 (31·1%) interpretable secY sequences and these were identical to those obtained from direct DNA sequencing of the urine samples from which they were recovered. Phylogenetic analyses identified four species: L. interrogans, L. borgpetersenii, L. noguchii, and L. santarosai with the most prevalent genotypes being associated with L. borgpetersenii. While direct sequencing cannot, as yet, replace culturing of leptospires, it is a valid additional tool for epidemiological studies. An unexpected finding from this study was the genetic diversity of Leptospira infecting Brazilian cattle.

Usage of a selective media (EMJH-STAFF) in primary culturing of pathogenic leptospires from bovine clinical samples.

UNLABELLED: Isolation of local strains is mandatory for the success of control programs. However, clinical samples are typically contaminated by other bacteria, which impair leptospires growth. The purpose of this study was to evaluate the use of a previously reported EMJH-STAFF media in the recovery of pathogenic leptospires from bovine clinical samples, namely urine (n = 123) and vaginal fluid-VF (n = 102). EMJH-STAFF presented less contamination than EMJH (<0·005), which was more evident in VF culture tubes. Nine pure leptospires cultures were obtained, six from urine (4·9%) and three from VF (2·9%). From those, seven grew on EMJH-STAFF, one on EMJH and one in both media. All the isolates were confirmed as pathogenic leptospires by lipL32-PCR, and sequencing of partial rrs showed them to belong to Leptospira noguchii, Leptospira santarosai and Leptospira interrogans species. EMJH-STAFF media was an important tool in the recovery of leptospires from bovine clinical samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The slow growth of leptospires and overgrowth of co-existing micro-organisms from environmental and microbiota are the major difficult to recovery Leptospira from animal clinical samples. Implementing an efficient control programme is essential to determine circulating leptospires in the region and their reservoirs. This study evaluated the relationship of a selective media (EMJH-STAFF) on the recovery of pathogenic leptospires (Leptospira noguchii, Leptospira santarosai and Leptospira interrogans), from bovine clinical samples (urine and vaginal fluid). EMJH-STAFF seems to be an important tool in obtaining local strains for epidemiological and control purposes.

First isolation of Leptospira noguchii serogroups Panama and Autumnalis from cattle.

Prevention and control of leptospirosis are based on the knowledge of locally circulating strains. Thus, efforts to obtain local isolates are paramount to the epidemiological understanding of leptospirosis. We report and discuss here the first isolation of members of serogroups Autumnalis and Panama from cattle, both belonging to Leptospira noguchii species. Urine samples (n = 167) were collected directly by puncture of the bladder from randomly selected cows from a slaughterhouse in Rio de Janeiro, Brazil, for bacteriological culture. Isolates were characterized by serogrouping and sequencing (rrs and secY genes). Overall, 10/167 positive urine samples (6%) were obtained. Sequencing of amplicons targeting for both rrs and secY genes identified two of them (2013_U73 and 2013_U232) as L. noguchii. Serogrouping of those strains indicated that 2013_U73 belonged to the Panama serogroup (titre 1600), and 2013_U232 to the Autumnalis serogroup (titre 12800). Both Panama and Autumnalis are known agents of incidental leptospirosis in cattle. This group of leptospires could be particularly important in tropical countries. This is the first report of members of serogroups Autumnalis and Panama belonging to L. noguchii species from cattle. Although related to previously reported strains, these isolates have been shown to be genetically diverse from them.