Vanessa Castro

Leptospira spp detection by Polymerase Chain Reaction (PCR) in clinical samples of captive black-capped Capuchin monkey (Cebus apella)

Leptospirosis is a widely distributed zoonosis that affects domestic and wild animals, and that has the man as the end point of its epidemiological chain. Leptospirosis diagnosis in primates is more difficult than in other animal species, as clinical signs and lesions are less evident and antibody response is detected only for short periods. The aim of this article was to describe the detection of Leptospira spp using polymerase chain reaction (PCR), in clinical samples from one captive black-capped Capuchin monkey (Cebus apella), which presented characteristics compatible with leptospirosis (jaundice and haemorrhagic kdney) in the macroscopic post-mortem examination. A friable kidney fragment and urine sample were cultured and submitted to experimental inoculation in guinea pigs and PCR using genus specific primer pair targeting the 16S rRNA region from Leptospira interrogans serovar canicola. Isolation of the agent was negative both in culture and experimental inoculation. The PCR amplification of the clinical samples showed a 330 pb amplified fragment that corresponds to the Leptospira genus. Based on these results PCR was considered an important tool for leptospira detection in nonhumam primates, more sensitive and specific than other techniques, especially considering that the viability of the pathogen was not possible. These advantages enable the detection of the leptospiras in urine and kidney, even when autolysed, frozen or badly conserved, which prevented the isolation and experimental inoculation from positive results.

An in vitro growth inhibition test for measuring the potency of Leptospira spp. Sejroe group vaccine in buffaloes

Leptospira spp. serovars Hardjo and Wollfi from Sejroe serogroup have been detected in livestock in Brazil, where the main control procedures rely on vaccination. The potency of two commercial vaccines available in this country was monitored by microagglutination test-MAT and in vitro growth inhibition test-GIT in serum samples from 33 female buffaloes divided into: G1-unvaccinated control; G2-vaccinated with Leptobac-6 containing serovars Hardjo and Wolffi and G3-vaccinated with Triangle-9 containing serovar Hardjo. G2 and G3 animals were vaccinated on day zero, and received a booster and two revaccinations on days 30, 210 and 390 and G1 animals received phosphate buffered saline. Serum samples were collected at 15-day intervals between days 0 and 60; and at 30-day intervals between days 60 and 540 and were tested by MAT and GIT with serovars Hardjo and Wolffi. G1 remained negative throughout the experiment. Both vaccines were able to induce agglutinating and growth inhibition antibodies. Six months after the last revaccination, all animals tested negative by MAT, but still were positive by GIT until the end of experimental period. GIT could be a good tool to evaluate the potency and to monitor antibodies responses of vaccines of Sejroe group serovars.

Leptospirose bovina no Estado da Paraíba: prevalência e fatores de risco associados à ocorrência de propriedades positivas

Herd-level and animal-level prevalences were determined and risk factors associated with leptospirosis were identified in cows in the State of Paraiba, Northeastern Brazil. A total of 2,317 cows with ≥ 24 months of age from 450 herds were randomly sampled. For the serological diagnosis of Leptospira spp. infection, the microscopic agglutination test (MAT) was carried out using 24 serovars as antigens. A herd was considered positive when presented at least one seropositive animal. Of the 450 investigated herds, 398 (89.7%; 95% CI = 86.6-92.2%) presented at least one reactant animal at MAT to any serovar, and 1,349 (61.1%; 95% CI = 56.6-65.4%) animals were seropositive. Serovar Hardjo was the most prevalent in herds and animals, with frequencies of 58.17% and 54.69% respectively. Location of the herd in the Sertao (odds ratio = 3.20; p=0.003), presence of wildlife (odds ratio =2.89; p=0.005), not cooling milk (odds ratio =3.83; p=0.034) and presence of flooded pastures (odds ratio =2.36; p<0.001) were identified as risk factors for herd-level prevalence. It is concluded that leptospirosis is widely spread in cattle in State of Paraiba, which reinforces the need for increased prevention and control measures, such as vaccination of herds. According to the results of risk factors analysis it is suggested that the sanitary control before the introduction of animals, drainage of wetlands and improvement in management conditions are important measures to preventing the infection.

Excretion of Brucella abortus vaccine B19 strain during a reproductive cycle in dairy cows

This paper aimed to determine the excretion period of B19 vaccine strain during a complete reproductive cycle (from estrus synchronization, artificial insemination, pregnancy and until 30 days after parturition) of dairy cows from 3 to 9 years old that were previously vaccinated from 3 to 8 months. Three groups were monitored with monthly milk and urine collection during 12 months: G1 with seven cows from 3 to 4 years old; G2 with three cows from 5 to 6 years old; and G3 with four cows from 7 to 9 years old. Urine and milk samples were submitted to bacteriological culture and urine and PCR reactions for detection of Brucella spp. and PCR-multiplex for B19 strain identification. Ring test (RT) was also performed in the milk samples, and serum samples were tested by buffered acidified plate antigen test (BAPA). All animals were serologically negative at BAPA and Brucella spp. was not isolated from both urine and milk samples. RT revealed 13/210 (6.2%) positive milk samples. PCR reactions detected DNA of Brucella spp. in 86/420 (20.5%) samples. In urine it was found a significantly higher frequency (35.2%; 74/210) than in milk (5.7%; 12/210), more frequently from the estrus to 150 days of pregnancy and after parturition (6.7%; 10/150), and from 150 days of pregnancy to parturition (3.4%; 2/60), and they were all identified as B19 strain. In three groups, intermittent excretion of B19 strain was detected mainly in urine samples, which confirmed its multiplication and persistence in cows for until 9 years.

Interference of vaccinal antibodies on serological diagnosis of leptospirosis in vaccinated buffalo using two types of commercial vaccines

The vaccinal antibodies interference represents one of the Microscopic Agglutination test - MAT limitation in the animal leptospirosis serum diagnosis. Prospective studies showing the dimensions of this effect are rare in buffaloes. This study aimed to determine the anti-Leptospira serum agglutinin profile in vaccinated female buffaloes using two types of commercial vaccines against leptospirosis: bacterin (whole bacterial cell) and purified outer membrane and to evaluate the vaccinal interference on serum diagnosis. Three groups of 11 adult buffalo females were established: G1-control, non-vaccinated, G2- vaccinated with bacterin vaccine with six serovars, G3- outer membrane purified vaccine with five serovars. A booster dose was administrated 30 days after the first vaccination (dpv) and two re-vaccinations six months a part (210 and 390 dpv). Serum samples were collected on days 0, 15, 40, 45, 60 and every 30 days until 540 dpv. G1, G2 and G3 serum samples were submitted to MAT with the serovars present in the vaccines. G1 remained always negative. Both vaccines induced serologic responses in MAT at 150 dpv against all serovars and they revealed maximum titers around 45 and 60dpv as follows: Pomona: G2 (1600) and G3 (3200); Hardjo: G2 and G3 (1600); Wolffi: G2 (800) and G3 (1600); Icterohaemorrhagiae: G2 and G3 (800); Grippotyphosa: G2 and G3 (200) and Canicola: G2 (NR) and G3 (400). Even though, the Wolffi serovar is not present in the purified outer membrane vaccine, G3 showed a response to that serovar, probably due to cross reaction to the serovar Hardjo. The G3 titers were higher and appeared earlier than in G2, but with similar serologic profiles. At the re-vaccination there was an increase on agglutinin levels, but of less intensity than those previously observed. After six months from the second revaccination (540 dfv), G2 and G3 were almost negative, which demonstrated the short diagnostic interference.

Seroprevalence and risk factors of antibodies against Leptospira spp. in ovines from Uberlândia municipality, Minas Gerais state, Brazil

The objectives of the present study were to verify the seroprevalence of anti-Leptospira spp. antibodies, identify the most frequent serovars and the risk factors associated with the infection in Uberlândia, Minas Gerais, Brazil . A total of 334 ovines blood samples were collected in 12 farms from Uberlândia municipality to be evaluated by means the Microscopic Agglutination Test (MAT) against 22 serovars of Leptospira spp. and an epidemiologic questionnaire was applied for each farm in order to correlate with risk factors of leptospirosis: sex, age and breed as well as contact with cattle, contact with dogs and presence of rodents. The prevalence of seropositive to MAT was found in seventy four ovines (22.2%; CI 95% 17.6-26.4%), with titers ranging from 100 to 3200. The most frequent serovars identified were: Hardjo, Autumnalis, Hardjo and Wolffi association and Grippotyphosa. Statistically significant differences were found in males, pure breeds and presence of rodents (p<0.05). The prevalence of anti-Leptospira spp. antibodies found in the present study demonstrated that this bacterium occurs in ovines of Uberlândia municipality, MG, Brazil . The need for the adoption of efficient management for the control of rodents and infection in ovines in order to avoid leptospirosis in the local flocks and future transmission to humans.

Prevalence and risk factors associated with Leptospira spp. infection in technified swine farms in the state of Alagoas, Brazil: risk factors associated with Leptospira spp. in swine farms.

The aim of this study was to determine the prevalence and to identify the risk factors associated with Leptospira spp. infection in technified pig farms in the state of Alagoas, Brazil. To compose sample for the prevalence study, 342 pigs were used (312 sows and 30 boars) proceeding from seven swine farms distributed in five districts of the state of Alagoas, Brazil. The infections serological diagnosis was performed by microscopic agglutination test. The risk factors analysis was performed using research questionnaires consisting of objective questions related to the breeder, the general characteristics of the property, and the productive, reproductive and sanitary management. Prevalence of 16.1% (55/342) of pigs seropositive was obtained. The associated risk factors were not performing quarantine (P = 0.003, OR = 5.43, CI = 1.79-16.41) and the use of artificial insemination (P = 0.023, OR = 3.38, CI = 1.18-9.66). A significant association of sow infection with the increased number of stillborn and mummified foetuses was found, as well as with the increased frequency of oestrus recurrence and the increased weaning-to-oestrus interval of seropositive sows. One might state that Leptospira spp. infection is disseminated in technified pig farms in the State of Alagoas, favouring reproductive failures and the impairment of zootechnical performance in these properties. The risk factors identified in this study are facilitators in the infecting agent dissemination and should be adjusted to control the disease in the herds studied.

Occurrence of antibodies anti -Toxoplasma gondii, Neospora caninum and Leptospira interrogans in a captive deer herd in Southern Brazil

A large number of Brazilian zoos keep many endangered species of deer, however, very few disease surveillance studies have been conducted among captive cervids. Blood samples from 32 Brazilian deer (Blastocerus dichotomus, Mazama nana and Mazama americana) kept in captivity at Bela Vista Biological Sanctuary (Foz do Iguaçu, Brazil) were investigated for 10 ruminant pathogens, with the aims of monitoring deer health status and evaluating any potential zoonotic risk. Deer serum samples were tested for Brucella abortus, Leptospira (23 serovars), Toxoplasma gondii, Neospora caninum, bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, foot-and-mouth disease virus, western equine encephalitis virus, eastern equine encephalitis virus and Venezuelan equine encephalitis virus. Antibodies against T. gondii (15.6%), N. caninum (6.2%) and L. interrogans serogroup Serjoe (3.1%) were detected. The serological results for all other infectious agents were negative. The deer were considered to be clinically healthy and asymptomatic regarding any disease. Compared with studies on free-ranging deer, the prevalences of the same agents tested among the captive deer kept at the Sanctuary were lower, thus indicating good sanitary conditions and high-quality management practices at the zoo.

Seroprevalence and risk factors for leptospirosis in cattle, sheep, and goats at consorted rearing from the State of Piauí, northeastern Brazil.

Leptospirosis is an endemic disease in Latin America, caused by pathogenic bacteria of the genus Leptospira. It is considered one of the main causes responsible for the negative economic impact on global livestock by causing reproductive problems. The research aimed to determine the prevalence of leptospirosis in cattle, sheep, and goats at consorted rearing in the micro-region of Teresina, Piauí state, northeastern Brazil, as well as to identify prevalent serovars and risk factors associated with seroprevalence. Serum samples were analyzed in 336 sheep, 292 goats, and 253 cattle using microscopic agglutination test (MAT). Overall, 378 samples were positive to MAT, with seroprevalence of 42.9%. The prevalences in cattle, sheep, and goats were 50.5, 40.5, and 34.6%, respectively. All herds presented at least one seropositive animal; the Hardjo/Wolffi serovar association was the most common in cattle and Icterohaemorrhagiae in goats and sheep. Beef production (OR = 4.9), cattle herd over 35 animals (OR = 4.0), feeding on pasture (OR = 6.4), weir and/or stream as water source (OR = 2.1), and no veterinary services (OR = 2.9) were risk factors for cattle infection. For sheep, intensive management system (OR = 5.3), suspended slatted facilities (OR = 2.2), more than 20 sheep in reproductive age (OR = 1.9), and absence of deworming (OR = 3.5) were the risk factors, while for goats, the identified risk factors were sheep herd over 52 animals (OR = 1.9) and no veterinary services (OR = 1.8). We conclude that the infection was spreading in consorted herds in this region. Thus, it would be interesting and important to conduct educative activities to farmers on the economic impacts of this disease and the need of preventive and control strategies mainly focused on sanitary measures and animal handling.

Risk factors to leptospirosis in cows in São Paulo state, Brazil

Risk factors associated to leptospirosis were identified in cows from Sao Paulo State. The State was divided into seven productive circuits from which 8,216 cows older than 24 months of age from 1,021 herds were sampled. For serological diagnosis of Leptospira spp. infection, the microscopic agglutination test (MAT) was carried out. Of the investigated herds, 718 (71.3%; 95% CI = 68.5% – 74.0%) presented at least one reactant animal at MAT to any serovar. Serovar Hardjo was the most prevalent, with 55.2% (95% CI = 51.4% 58.9%) of the positive herds. Herd size, animal purchase, share pasture, presence of ovine and swine, and utilization of artificial insemination were identified as risk factors. Utilization of maternity pens was a protective factor against leptospirosis.