Camila Hamond

Leptospirosis as the most frequent infectious disease impairing productivity in small ruminants in Rio de Janeiro, Brazil.

Despite the importance of small ruminants breeding in developing countries, milk/meat productivity remains unsatisfactory. Infectious diseases, such as leptospirosis, brucellosis, and small ruminant lentiviruses (SRLVs), contribute to this scenario. The objective of the present study was to determine the role of each of these diseases in the productivity of small ruminants breeding in Rio de Janeiro, Brazil. In goats, 343 samples were tested for leptospirosis, 560 for Brucella abortus, and 506 for caprine arthritis-encephalitis (CAE), whereas in sheep, 308 samples were tested for leptospirosis, 319 for B. abortus, 374 for Brucella ovis, and 278 for Maedi-Visna (MV). Regarding leptospirosis, 25.9% of goats and 47.4% sheep were seroreactive, with serovar Hardjo the most prevalent in both species. Anti-B. abortus agglutinins were found in 0.7% of all samples, exclusively in goats. In relation to SRLVs, 8.6% of goats and 3.2% of sheep samples were positive for CAE and MV, respectively. Leptospirosis was the major infectious problem in the small ruminants sampled and may contribute to impaired productivity of these animals.

The role of horses in the transmission of leptospirosis in an urban tropical area.

The objective of this study was to demonstrate the presence of leptospires in equine urine, as evidence for a potential role of horses in transmission of this organism. Thoroughbred horses (aged 2-5 years, n = 276) from Rio de Janeiro, Brazil, were studied. After a severe storm, the premises of the animals remained flooded for 72 h. Blood samples for serology were collected on days 20 and 35 (day of storm = day 0). On day 20, 132 (47·8%) horses were seroreactive (titre ≥200) and, of these, 23 (31·0%) had increased antibody titres on day 35. Furthermore, 34 urine samples (for PCR and culture) were collected from seroreactive horses on day 35. Copenhageni was the most frequent serovar (88·8% of reactive titres). Although none of the urine samples were culture positive, 12 (35·2%) were PCR positive. This is apparently the first report of evidence of leptospires in urban horses. Furthermore, we suggest that these animals can play a role in the transmission of leptospirosis in urban areas.

Serology and PCR for bovine leptospirosis: herd and individual approaches

LEPTOSPIROSIS is an important zoonotic bacterial infection of livestock that causes abortions, stillbirths, infertility and reduction of milk production, determining economic hazards. Cattle act as the maintenance host of serovar Hardjo (serogroup serjoe), which is the most common strain of Leptospira in many rural regions (Hernandez-Rodriguez and others 2011). The standard serological diagnosis of leptospirosis is the microscopic agglutination test (MAT), which is recommended for use on a herd-screening basis (Lilenbaum and others 2009). Since leptospiral cultivation is laborious and time-consuming, other methods have been applied for the direct identification of leptospires (Marianelli and others 2007). PCR is increasingly being reported as a rapid and definitive diagnostic tool in many species, such as buffaloes (Marianelli and others 2007), small ruminants (Lilenbaum and others 2009) and cattle (Hernandez-Rodriguez and others 2011). The aim of the present study was to compare the use of MAT and PCR for the diagnosis of leptospirosis in cattle. Three dairy herds (A, B and C) from Rio de Janeiro, Brazil, with high levels of reproductive failure, …

The role of leptospirosis in reproductive disorders in horses

Leptospirosis is a zoonotic disease of global importance and has a worldwide distribution. This infection displays clear seasonal nature in some regions of the tropics, where the rainy season is marked by high temperatures. Household and wild animals carry leptospires and contribute to their dissemination in nature. Transmission mainly occurs by contact with water contaminated with the urine of infected animals, and consequently, it is quite widespread especially in times of rain, since many areas are subject to flooding and have poor sanitation. Serological tests demonstrate that Leptospira sp. infection in horses occurs worldwide and that the predominant serovar may vary depending on the region or infection sources. Besides systemic and ocular manifestations, leptospirosis in horses has been recognized as an important disease of the reproductive system, since it leads to the birth of weak foals, stillbirths or neonatal mortality, and mainly to abortion, usually after the sixth month of pregnancy. In this context, this review aims to gather and discuss information about the role of leptospirosis in reproductive disorders in horses.

Diagnosis and control of an outbreak of leptospirosis in goats with reproductive failure

This study presents a Brazilian goat herd with reproductive failure over 2009-2010, in which there were abortions (22/50; 44%), embryonic resorption (6/50; 12%) and neonatal deaths (2/50; 4%). A diagnosis of leptospirosis was made, based on serology (microscopic agglutination test - MAT), bacterial culture, and polymerase chain reaction (PCR). Antibiotic therapy, specific vaccination protocols and changes in management practices were instigated. One year after the outbreak, diagnostic methods were repeated and reproductive performance re-analysed. Soon after the outbreak, 61/125 (48.8%) of the goats were seropositive for Leptospira. Pure isolates of Leptospira were not obtained, but Leptospira PCR testing was positive in 48/50 (96%) urine samples. After 1 year only 4.2% were seropositive and the occurrence of reproductive problems decreased roughly 10-fold, although five goats (10.4%) remained PCR-positive. A broad-based management approach, including serological and molecular diagnostic methods, vaccination, antibiotic treatment, and alteration of some environmental aspects, were critical to the control of this outbreak, thereby minimising subsequent reproductive failures and economic losses.

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.

A multilocus variable number tandem repeat analysis assay provides high discrimination for genotyping Leptospira santarosai strains.

Considering the prevalence of Leptospira santarosai infections in the Americas and the scarce information about the species, we aimed to apply a multilocus variable number tandem repeat (VNTR) analysis (MLVA) for the molecular typing of L. santarosai isolates from various sources. Amplification of three VNTR loci selected from L. santarosai genome sequences resulted in a wide range of sizes for the amplified products amongst the 21 L. santarosai strains analysed. This suggested a variation in tandem repeat copy numbers in the VNTR loci. secY sequencing also showed a high nucleotide diversity, confirming the MLVA data. In conclusion, this novel MLVA provided a high level of discrimination between L. santarosai isolates, and this new typing tool could be used to investigate leptospirosis in regions where L. santarosai predominates.

Infection by Leptospira spp. in cattle in a tropical region, Rio de Janeiro, Brazil.

Dear Sir: We read with interest the study reported by Ayral and others1 entitled “Distribution of Leptospira serogroups in cattle herds and dogs in France.” Ayral and others1 concluded that the inventory of infecting Leptospira serogroups revealed that current vaccines in France are not fully capable of preventing leptospirosis. In total, 394 cattle were diagnosed with clinical leptospirosis, and the results suggested infection by serogroups Australis (43%), Sejroe (33%), and Grippotyphosa (17%). We share our experience with bovine leptospirosis in Brazil and compare it with the scenario described in France. Infected cattle are commonly asymptomatic and may shed the bacterium in urine for long periods.2 Serology by microscopic agglutination test (MAT) is recommended as the primary diagnostic tool, although it is considered reliable only at the serogroup level.3 Although useful for a diagnosis at the herd level, serology is not adequate for the individual detection of carriers,4 which may impair control programs.5 For individuals, a rapid, sensitive, and reliable diagnostic tool, such as polymerase chain reaction (PCR), is required. In a study conducted by our group in 2013, 208 cows were randomly selected in a slaughterhouse located close to Rio de Janeiro, Brazil. After clinical pre-slaughter examination, the animals were considered free of signs of leptospirosis. Blood samples (208) for serology (MAT)3 and urine samples (199) from direct puncture of the bladder for PCR-lipL325 and culture were obtained. Serology showed that 77 of 208 (37%) animals were reactive, with serogroup Sejroe by far the most frequent (62.3%) followed by Javanica (7.8%), Icterohaemorrhagiae (6.5%), Grippotyphosa (3.9%), and Tarassovi (3.9%). Urine PCR showed that 67 of 199 animals (33.6%) were positive. Additionally, 10 isolates were obtained in pure culture, including serogroups Sejroe, Shermani, and Grippotyphosa with 2 isolates each and Sarmin, Tarassovi, Autumnalis, and Panama with 1 isolate each. These results showed a high rate of asymptomatic shedding in cows. Members of serogroups Grippotyphosa, Autumnalis, and Panama are usually associated with environmental contamination and maintained by wildlife species. Serogroup Sejroe is the most common in ruminants worldwide as well as in Brazil.6 Serogroups Sarmin, Shermani, and Tarassovi have never previously been reported from cattle in Brazil and may have been neglected as agents of bovine leptospirosis. Serogroup distribution differed between Brazil and France. In our study, both seronegative and seroreactive cows were shedding leptospires. Therefore, the direct detection of leptospires in urine by culturing or PCR is an important tool for the success of control programs in cattle. Additionally, an active surveillance approach should also include asymptomatic animals, because they may shed the bacterium to the environment and consequently, play an important role in the epidemiology of leptospirosis.

Pulmonary hemorrhage in horses seropositive to leptospirosis

The aim of this study was to verify the occurrence and severity of pulmonary hemorrhage (PH) in horses seropositive or not to leptospirosis. A total of 84 training horses were tested for anti-Leptospira antibodies and submitted to endoscopy of the upper airways 30-60 minutes after exercise. Forty-nine (46.4%) horses were seropositive (titer>100) to serovar Copenhageni. Seventeen horses (20.2%) presented PH in different grades, significantly more commonly observed in seropositives (34.7%) than seronegatives (8.6%) (p<0.05), and also more severe in the first. Additionally, a seropositive horse is 4.26 times more likely (OR) to present PH than a seronegative one. Fifteen days after specific antibiotic treatment for leptospirosis a significant improvement in grade of hemorrhage was observed. Therefore it is suggested that leptospirosis may potentiate the severity of PH and that specific antibiotic treatment directed towards leptospirosis may help to control the disease.

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.