Ana Paula Loureiro

Isolation of Leptospira interrogans Hardjoprajitno from vaginal fluid of a clinically healthy ewe suggests potential for venereal transmission.

A total of 15 adult ewes from one flock known to be seroreactive for leptospirosis was studied. Urine and vaginal fluid were collected from each animal to test for the presence of leptospires using bacterial culture and conventional PCR methods. One pure culture of Leptospira sp. was obtained from the vaginal fluid sample of a non-pregnant ewe. The isolate was characterized by DNA sequencing of the rrs and secY genes, variable-number of tandem-repeats (VNTR) analysis and serogrouping, and the isolate was typed as Leptospira interrogans serogroup Sejroe serovar Hardjo type Hardjoprajitno. This report indicates the presence of viable Leptospira in the vaginal fluid of a ewe, suggesting the potential for venereal transmission of leptospires in sheep.

Usage of Leptospira spp. local strains as antigens increases the sensitivity of the serodiagnosis of bovine leptospirosis.

Leptospirosis is a zoonotic disease that occurs worldwide, particularly in tropical countries. In livestock the agent is responsible for reproductive problems such as infertility and abortion. Serogroup Sejroe, particularly serovar Hardjo, prevails in cattle in several regions. The microscopic agglutination test (MAT) is the current method for diagnosing leptospirosis. It has been proposed that the inclusion of local strains could detect a larger set of seroreactive animals. In that context, the aim of the present study was to evaluate if the usage of local strains as antigens increases the sensitivity of the serodiagnosis of bovine leptospirosis. Blood and urine samples were collected from 314 bovines from several herds randomly selected in a slaughterhouse in Rio de Janeiro, Brazil. Serological diagnosis was made with MAT using a 21 reference-strains panel (MAT21). Additionally, 12 local strains (MAT33) were included as antigens. PCR was performed with the urine samples and it was positive on 71 out of 222 samples (31.9%). MAT21 identified as seroreactive 173 (55.1%) out of the 314 animals studied, with Sejroe the most common (38.1%). In MAT33, 204 (65.0%) animals were seroreactive with a significant increase on seroreactivity (9.9%). In conclusion, MAT presented with a significant increase of sensitivity when local strains were used as antigens. Among the local strains, 2013_U152 (KP263062) (serogroup Shermani) and 2013_U280 (KP263069) (serogroup Grippotyphosa) showed to be more antigenic.

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.

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.

Effects of rainfall on incidental and host-maintained leptospiral infections in cattle in a tropical region

This study investigated the effects of rainfall on incidental and host-maintained leptospiral infections in cattle in a tropical region. Serum and urine specimens were collected from 582 bovines in a slaughterhouse. Seropositivity during rainy seasons was 43.6% (158/362) and during dry seasons was 31.8% (70/220; P = 0.0047). Positivity by urine PCR in rainy seasons was 42.3% (153/362) and 33.2% (73/220; P = 0.0296) in dry seasons. Additionally, increases in rainfall rates had different effects on host-adapted (serogroup Sejroe) and incidental (all other serogroups) leptospiral infections. There was a significant increase in the number of cattle shedding Leptospira during the rainy season (CI 1.04-2.09; P = 0.0296), suggesting temporary (transient) urinary shedding of incidental serogroups. Incidental and host-maintained Leptospira infections in cattle require different diagnostic techniques and control strategies.

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 and serological characterization of the first Leptospira santarosai strain isolated from a dog

Leptospirosis is a zoonotic disease of global importance caused by pathogenic Leptospira species. Dogs can become asymptomatically infected, acting like reservoir hosts for pathogenic Leptospira, notably Leptospira interrogans serovar Canicola. Identification of such individuals and characterization of leptospires involved in chronic infections may unravel the role of dogs in the epidemiology of particular leptospiral strains. The aim of the present work was to describe the first Leptospira santarosai strain isolated from a dog. The dog was kept in a public shelter in São Paulo city, Brazil, and presented asymptomatic urinary shedding detected by PCR. Prospective evaluation was performed to fully characterize its chronic carrier state. The dog did not present anti-Leptospira titles or clinical/laboratorial abnormalities during the evaluations; nevertheless long-term urinary shedding was confirmed by PCR and leptospires were recovered from two occasions. The isolated strain was molecularly characterized by partial 16S rRNA and secY gene sequencing and MLST analysis. Serogroup identification was performed using polyclonal antibodies. The strain was identified as Leptospira santarosai, serogroup Sejroe. This is the first evidence in the literature of the isolation of L. santarosai in dogs. Our findings show that dogs can persistently harbor leptospires other than L. interrogans.

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

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.

First isolation and characterization of Leptospira interrogans serogroup Australis from swine in Brazil

The purpose of this study was to report the first recovery and characterization of Leptospira interrogans (serogroup Australis) from urine of swine in Brazil. The isolate was studied by serogrouping, MLVA, PGFE, and partial sequencing of rrs and secY. It was serogrouped as serogroup Australis, probably serovar Bratislava (titre 1,600), and sequenced as Leptospira interrogans. The MLVA and PGFE profiles also suggested the isolate as serovar Bratislava, since they were indistinguishable from reference strains Balico and Jez Bratislava. This is the first Leptospira interrogans serogroup Australis isolate, probably serovar Bratislava, obtained in Brazil.

Detecção molecular de ovinos carreadores de Leptospira em ambiente tropical

Braz. J. Vet. Res. Anim. Sci., São Paulo, v. 51, n. 3, p. 220-223, 2014 Abstract The purpose of this study was to analyze the usefulness of PCR for the detection of leptospiral carriers in sheep under tropical field conditions. Two flocks, previously reported as seroreactive (A) and seronegative (B), were selected for this study. From those, the totality of animals of each flock, urine and vaginal fluid (VF)/semen were collected for bacteriological culture and PCR, as well as serum samples for serology. Serology confirmed the previous status of the two flocks. Culture was negative for all the samples. In PCR, animals of Flock A presented 26.7% (VF), 33.3% (semen) and 38.9% (urine) of positivity. Flock B presented 40.0% (VF), 33.3% (semen) and 5.6% (urine) of positivity by PCR. In conclusion, PCR was important to identify carriers of leptospires, including animals from a seronegative flock, what reinforces the advantages of the usage of this tool for the detection of carriers in sheep as part of control programs of leptospirosis under tropical field conditions.