Carriers Of Leptospires Research Articles

Detecting Leptospira spp. infection in cows by PCR: What is the best sample to test?

Bovine leptospirosis is a major reproductive disease. As cows can be leptospiral carriers both on the renal and genital tract, diagnosis can be challenging, with an underlying risk of misdiagnosis. Traditionally, the infection has been diagnosed by culturing or PCR from urine samples. Nevertheless, recent studies have suggested testing genital samples rather than urine, particularly for the diagnosis of genital colonization and reproductive disorders. The present study aimed to compare urine versus genital samples to detect leptospiral carriers in naturally infected cows with poor reproductive performance under field conditions. Five herds presenting >20 % of seroreactive animals against the Sejroe serogroup were selected. Of these, 106 cows with poor reproductive performance were studied, and urine, uterine fragment (UF), and cervicovaginal mucus (CVM) were obtained and tested by lipL32-PCR. A total of 73 (68.9 %) cows were infected; 64 of which (87.7 %) were diagnosed via positive genital samples (UF and/or CVM), while only 14 (19.2 %) by urine (p ≤ 0.001). Therefore, if the study had been limited to urine samples, as largely recommended, less than 20 % of the infected cows would have been detected, representing a huge misdiagnosis of the disease that could undermine the efficacy of control programs. In this context, the present study reinforces prior findings that testing genital samples, particularly CVM, is crucial to effectively diagnosing infected subfertile cows.

Leptospirose genital bovina: achados em touros mantidos em condições do bioma Caatinga

ABSTRACT: Leptospirosis is a disease that causes economic and social impact, as it affects wild and domestic animals and humans. There may be peculiarities in the epidemiology of this disease in the Caatinga biome, Brazil, where the environment is adverse and the etiologic agent, Leptospira spp., requires alternative transmission routes. Considering that in bovine leptospirosis the genital carrier is constantly neglected and the lack of reports on the role of bulls in the epidemiology of the bovine genital leptospirosis (BGL) syndrome, mainly in semiarid conditions such as Caatinga biome, this study aimed to investigate bulls maintained in Caatinga biome conditions as genital carriers of leptospires. Urinary tract (urine, bladder, and kidney) and reproductive tract (vas deferens, cauda epididymis, and vesicular gland) samples were collected from 42 slaughtered bulls. Microscopic agglutination test (MAT), polymerase chain reaction (PCR), and microbiological isolation were included as diagnostic methods. Anti-Leptospira spp. antibodies were found in 17 (40.48%) animals, while 26 animals (61.90%) had at least one organ or urine with leptospiral DNA, and 10 animals (23.81%) were positive at bacteriological culture. Sequenced samples targeting the LipL32 gene showed 99% similarity with Leptospira borgpetersenii. Molecular analysis of samples from the vas deferens and cauda epididymis is recommended for the diagnosis of genital leptospirosis in bulls and, if it is impossible to collect these tissues, semen can be used. In conclusion, this study provides important information relating to bulls from the Caatinga biome, Brazil, as carriers of genital leptospirosis. The results indicate that, even under adverse environmental conditions, leptospires may survive and propagate, mainly due to the characteristic of genital carriers for the sexually spreading of adapted Leptospira species without influence by external variables. Thus, prevention and control strategies for bovine leptospirosis need to include actions aimed at the genital carrier.

Persistent High Leptospiral Shedding by Asymptomatic Dogs in Endemic Areas Triggers a Serious Public Health Concern.

Simple SummaryDogs are known as hosts of Leptospira interrogans and can spread this bacterium to the environment. Although Canicola is responsible for determining chronic disease in dogs, when affected by incidental serogroups such as Icterohaemorrhagiae, acute disease may occur with a predominance of clinical signs with hepatic and renal lesions. In endemic areas, it is a serious public health problem, as dogs can become asymptomatic carriers of leptospires in the urine, characterizing a risk in the context of zoonotic leptospirosis. Thus, this study aims to estimate the incidence and duration of the elimination of leptospires in the urine of dogs, taking another step from a previous study of our group, by a longitudinal, long-term and molecular approach. A total of 125 dogs without signs of leptospirosis were included in the study. Of the 125 dogs, 62 were PCR-positive (48.8% (95% CI, 47.9–49.7%)) throughout the study. Most dogs were shedding the Icterohaemorrhagiae serogroup in urine, which was unexpected, since the animals remained clinically asymptomatic during the study. Although the fact that asymptomatic dogs eliminate leptospires is not new, the extent of this fact and that the strain is virulent is impressive, with an impact on public health that cannot be overlooked.(1) Background: Dogs are known as hosts of Leptospira interrogans and can spread this bacterium to the environment. Although Canicola is responsible for determining chronic disease in dogs, when affected by incidental serogroups such as Icterohaemorrhagiae, acute disease may occur with a predominance of clinical signs with hepatic and renal changes. In endemic areas, it is a serious public health problem. Thus, this study aims to estimate the incidence and duration of elimination of leptospires in the urine of dogs, taking another step from a previous study of our group, by a longitudinal, long-term and molecular approach. (2) Methods: A total of 125 dogs without apparent symptoms of leptospirosis were included in the study. The dogs were all PCR-negative and seronegative at the beginning of the study. Blood samples were collected for hematological examinations and urine for amplification of the lipL32 gene by PCR at five different time points during one year. (3) Results: Out of the 125 dogs, 62 became lipL32 PCR-positive (48.8% (95% CI, 47.9–49.7%)) at some point during the study, distributed as follows: at day 0, all negative; day 90, 18/125 (14.4% (95% CI, 13.5–15.3%)); day 180, 18/125 (14.4% (95% CI, 13.5–15.3%)); day 270, 12/125 (9.6% (95% CI, 8.7–10.5%)); and day 365, 14/125 (11.2% (95% CI, 10.3–12.1%)). Out of the 62 amplicons, 22 were sequenced, targeting a short region of secY gene. Of these, 20 (90.9%) were identical to the L. interrogans serovar Icterohaemorrhagiae, while two (9.1%) were Leptospira noguchii. (4) Conclusions: The fact that the leptospires of the Icterohaemorrhagiae serogroup were characterized was unexpected, since the animals remained clinically asymptomatic during the study. The fact that asymptomatic dogs shed leptospires is not new, but the extent of this fact and the characterized strain is impressive, with an impact on public health that cannot be overlooked.

Neotropical wild rodents Akodon and Oligoryzomys (Cricetidae: Sigmodontinae) as important carriers of pathogenic renal Leptospira in the Atlantic forest, in Brazil

Leptospirosis is an important worldwide zoonosis, caused by a bacterium of the genus Leptospira. For a better understanding of the disease, it is relevant the application of the one health concept. The Atlantic Forest is considered a biodiversity hotspot, with a great endemism of species and despite its importance and proximity to urban areas, the potential role of its fauna as carriers of infectious agents is still poorly understood. Although it is well-known that rats and mice are key reservoirs of leptospires, particularly Rattus norvegicus, wild rodents have also been revealed as reservoirs of leptospiral strains. Thus, the purpose of this study was to investigate the role of the sigmodontine rodents from genera Akodon and Oligoryzomys as pathogenic Leptospira carriers in the Atlantic Forest. We studied 52 Akodon spp. and 15 Oligoryzomys spp. from three areas. Overall 30% were PCR-positive for pathogenic Leptospira, 27% (14/52) Akodon spp. and 40% (6/15) Oligoryzomys spp. DNA sequencing of LipL32 gene confirmed nine species as pathogenic Leptospira. This remarkable incidence of leptospiral carriage within wild genera emphasizes the role of these rodents as carriers of leptospires throughout in this environment.

Pathogenic Leptospira species are widely disseminated among small mammals in Atlantic Forest biome.

Leptospirosis is a common worldwide bacterial zoonosis and has been studied in One Health approaches. Small mammals are described as the most important maintenance reservoirs of several pathogens in nature, including leptospires. The aim of this study was to identify infection by leptospires among small mammals on the Atlantic forest biome and evaluate their potential as carriers of these spirochetes. A total of 153 small mammals belonging to orders Rodentia and Didelphimorphia (distributed on 17 genera and 22 species) were captured. Blood and kidney samples were collected from animals and a conventional PCR targeted on lipL32 gene was conducted on renal tissues. Species identification was performed in eight samples by sequencing of rrs gene. A total of 28% of the animals presented lipL32 PCR-positive, and four pathogenic Leptospira species (L.interrogans, L.borgpetersenii, L.santarosai and L.noguchii) were identified. This study highlights the role of small mammals as carriers of leptospires on the Atlantic Forest representing a potential source of pathogenic Leptospira spp infection for both humans and domestic animals.

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.

The role of capybaras as carriers of leptospires in periurban and rural areas in the western Amazon

Although leptospirosis has been described in capybaras, usually based on serological evidences, bacterial culture of leptospires has been scarcely reported in this species. The western Amazon is a reportedly endemic area where high seroprevalences have been reported in different species of wildlife, domestic animals and in human beings. The present study aimed at investigating the role of capybaras as carriers of leptospires in periurban and rural areas in the western Amazon region. A total of 44 animals were captured, and 41 blood samples (for serology) and 41 urine samples (for PCR and bacterial culture) were obtained. A total of 18/41 (43.9%) of sera were reactive and titers were generally low, indicating chronic infection. PCR was positive in 13/41 (31.7%) samples, isolates were recovered from urine samples belonging to Icterohaemorrhagiae, Grippotyphosa and Shermani serogroups. A high number of carriers (confirmed by PCR) associated to a tendency for harboring Icterohaemorrhagiae serogroup strains could be noticed. Our results suggest that capybaras are massively infected by leptospires. Analogously to Norway rats, capybaras present chronic infection with low titers and long-term bacterial shedding, and may be acting as reservoirs of this bacterium.

Detection of wild animals as carriers of Leptospira by PCR in the Pantanal biome, Brazil

Leptospiral infection is widespread in wildlife. In this context, wild ecosystems in tropical countries hold a vast biodiversity, including several species that may act as potential reservoirs of leptospires. The Pantanal biome presents highly favorable environmental conditions for the occurrence of leptospirosis, such as high temperatures, constant flooding, and high biodiversity. The purpose of this study was to detect wild animals as carriers of Leptospira sp. using direct methods (PCR and culture) in the Pantanal biome, Brazil. A total of 35 animals were studied, namely Cerdocyon thous, Nasua nasua, Ozotoceros bezoarticus, and Sus scrofa species. Blood for serology (MAT) and urine for bacteriological culturing and PCR was sampled. The most prevalent serogroups were Javanica and Djasiman. Additionally, 40.6% of these animals presented PCR positive reactions. Seroreactivity associated with the high frequency of leptospiral carriers among the different studied species suggests a high level of exposure of the studied animals to pathogenic Leptospira strains. Our results are still limited and the actual role of the studied animals in the epidemiology of leptospirosis in the Pantanal region remains to be elucidated.

Molecular detection of leptospiral carriers in sheep under tropical field conditions

O objetivo do presente estudo foi analisar a aplicabilidade da PCR na detecção de ovinos carreadores de Leptospira em ambiente tropical. Brevemente, dois rebanhos ovinos, previamente reportados como sororeativo (A) e soronegativo (B) foram selecionados para este estudo. Da totalidade de animais de cada rebanho, amostras de urina e fluido vaginal (FV)/sêmen foram colhidas para cultura bacteriológica e PCR. Além disso, amostras de soro foram colhidas e utilizadas na sorologia (teste da soroaglutinação microscópica). Esta técnica confirmou o estado prévio dos dois rebanhos. Nenhuma amostra pura de leptospiras foi obtida no cultivo. Já na PCR, animais do Rebanho A apresentaram 26,7% (FV), 33,3% (sêmen) e 38,9% (urina) de amostras positivas. O Rebanho B apresentou 40,0% (FV), 33,3% (sêmen) e 5,6% (urina) de positividade pela PCR. Em conclusão, a PCR foi uma importante ferramenta na identificação de carreadores de leptospiras, incluindo animais do rebanho soronegativo, o que reforça as vantagens do uso desta técnica para a detecção de ovinos portadores como parte dos programas de controle da leptospirose em ambiente tropical.

Urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock

The aim of the present study was to consider the wide usage of urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock. A total of 512 adult animals (300 cattle, 138 horses, 59 goats and 15 pigs), from herds/flocks with reproductive problems in Rio de Janeiro, Brazil was studied by serology and urinary PCR. From the 512 serum samples tested, 223 (43.5 %) were seroreactive (cattle: 45.6 %, horses: 41.3 %, goats: 34%and pigs: 60 %). PCR detected leptospiral DNA in 32.4 % (cattle: 21.6 %, horses: 36.2 %, goats: 77.4 % and pigs: 33.3 %. To our knowledge there is no another study including such a large number of samples (512) from different species, providing a comprehensive analysis of the usage of PCR for detecting leptospiral carriers in livestock. Serological and molecular results were discrepant, regardless the titre, what was an expected outcome. Nevertheless, it is impossible to establish agreement between these tests, since the two methodologies are conducted on different samples (MAT - serum; PCR - urine). Additionally, the MAT is an indirect method and PCR is a direct one. In conclusion, we have demonstrated that urinary PCR should be considered and encouraged as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock.

Leptospirosis in sheep and goats under tropical conditions

Inadequate management practices and poor reproductive performance have been reported as fundamental factors on reducing the levels of productivity in livestock. Different pathogens have been reported in small ruminants' herds/flocks with reproductive failures. The aim of the present study was to review aspects of leptospirosis in small ruminants, mainly its impact on reproduction and consequently on productivity of the herds/flocks under tropical conditions. Leptospiral infection in goats and sheep is common in several countries, and those species can also act as carriers of leptospires. Severe disease is often associated to young animals and is frequently associated to incidental serovars. In contrast, subclinical infection is mainly characterized by reproductive problems, such as infertility, abortion, occurrence of stillbirths, and weak lambs/goat kids. Moreover, laboratorial tests are essential to achieve an accurate diagnosis of the infection. Microscopic agglutination test is the most common indirect test of leptospirosis, being used worldwide. In small ruminants, PCR consists on a recommendable method for diagnosing animals that carry leptospires. Control of leptospirosis in small ruminants involves measures such as the identification and treatment of the carriers and other sources of infection, quarantine in acquired animals, and systematic immunization with commercial vaccines containing the circulating serovars in the herd/flock. Productivity of small ruminant breeding can dramatically increase with adequate sanitary conditions and control of leptospirosis. Immunization of all the animals combined to the treatment of carriers may successfully control the infection and importantly reduce the economic reproductive hazards that are observed under tropical conditions.

Molecular characterization of virulent Leptospira interrogans serogroup Icterohaemorrhagiae isolated from Cavia aperea

Leptospirosis is a worldwide zoonotic infection caused by pathogenic Leptospira. Synanthropic rodents are recognized carriers of leptospires; however, the role of wild rodents in the epidemiology of the disease is still incipient. In this work, we describe Leptospira strain isolated from Cavia aperea (Brazilian guinea pig). The isolated strain was characterized by partial rpoB gene sequencing, variable-number tandem-repeats and histopathological analysis. The strain was identified as Leptospira interrogans, serogroup Icterohaemorrhagiae and caused clinical signs of leptospirosis in the hamster model, attesting to its virulence. In conclusion, these findings could be useful for elucidating the epidemiological role of C. aperea in leptospirosis.

PCR detection of leptospiral carriers among seronegative horses

WE recently published a short communication about bovine leptospirosis demonstrating that, although serology was an acceptable screening tool for leptospirosis on a herd basis, it was not a good predictor...

Animal leptospirosis in small tropical areas

Leptospirosis is the most widespread zoonosis in the world. Humans become infected through contact with the urine of carrier animals, directly or via contaminated environments. This review reports available data on animal leptospirosis in ten tropical islands: Barbados, Martinique, Guadeloupe, Grenada, Trinidad, New Caledonia, Hawaii, French Polynesia, La Réunion and Mayotte. Leptospirosis is endemic in these insular wild and domestic fauna. Each island presents a specific panel of circulating serovars, closely linked with animal and environmental biodiversity, making it epidemiologically different from the mainland. Rats, mongooses and mice are proven major renal carriers of leptospires in these areas but dogs also constitute a significant potential reservoir. In some islands seroprevalence of leptospirosis in animals evolves with time, inducing changes in the epidemiology of the human disease. Consequently more investigations on animal leptospirosis in these ecosystems and use of molecular tools are essential for prevention and control of the human disease.

Pesquisa de suínos portadores renais de leptospiras pelo isolamento microbiano e reação em cadeia pela polimerase em amostras de rins de animais sorologicamente positivos e negativos para leptospirose

With the purpose to study the presence of swine leptospiral carrier,131 blood samples and the kidneys from the respective animals were taken during slaughter in abattoir house in Botucatu - SP region. By the Microscopic Agglutination Test (MAT), 48 serum samples were positive against one or more serovars, with a rate of the occurrence of 36.64% to anti-leptospiral antibodies and greater importance to the serovar icterohaemorrhagiae. The investigation of the agent, a total of 88 kidney samples were submitted to culture in EMJH media and analysed by PCR. From those 88 kidney samples, 48 were of serologically positive animals and 40 of negative animals, being isolated and detected the agent in one kidney sample, belonging to a seropositive animal. Although was not possible the statistical evaluation in terms of sensitivity and specificity of the two test to detection, the PCR showed faster and most practical to study leptospiral carriers. By the results of isolation it must be emphasized the importance of those animals as the transmitters of the disease to slaughter house workers and to meat inspectors.

Leptospirosis on animal reproduction: IV. Serological findings in mares from six farms in Rio de Janeiro, Brazil (1993-1996)

From February 1993 until October 1996, 547 blood samples were collected from mares with reproductive problems in six farms of the state of Rio de Janeiro, Brazil, and tested by the microscopic agglutination test (MAT) with live antigens for antibodies to seven leptospira serovars. A total of 235 positive reactions (42.96%) at a minimum serum dilution of 1:100 were observed against one or more serovars, which in comparison to other studies developed in Brazil, may be considered as a high prevalence. On mares with anti-leptospiral antibodies, the predominant serovar was icterohaemorrhagiae, reactive on 102 (43.40%) samples, followed by 64 (27.23%) reactions against bratislava, and 34 (14.47%) against pomona. The high prevalence of antibodies to serovar icterohaemorrhagiae observed in this study could have been expected, because in Brazil about 36% of the population of the maintenance host of such strain, Rattus norvegicus, are leptospiral carriers, mainly to strains of the Icterohaemorrhagiae serogroup.

Boars as carriers of leptospires of the Australis serogroup on farms with an abortion problem.

Boars as carriers of leptospires of the Australis serogroup on farms with an abortion problem.