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Molecular demonstration of intermittent shedding of Leptospira in cattle and sheep and its implications on control/Demonstracao molecular da intermitencia eliminando Leptospira em vacas e ovelhas e suas implicacoes no controle.

INTRODUCTION

Leptospirosis is a zoonotic disease of worldwide distribution, affecting both wildlife and domestic animals. In livestock it often presents as a silent and asymptomatic infection (DIRECTOR et al., 2014). Leptospirosis in livestock leads to reproductive failure, such as oestrus repetition, abortion, stillbirths and weak off spring (ELLIS, 2015). The real impact of these affections has not yet been estimated, but it is well known that these reproductive symptoms are related to economic losses (AYRAL, 2013).

Leptospires penetrate the host for lesions on the skin and mucous membranes. After penetration the bacteria invade the circulation (leptospiremia), spreading throughout the animal (ADLER, 2014). After this initial phase, it is known that leptospires lodges in the renal tubules of infected animals, being shed for long periods (leptospiruria), contaminating the environment and other animals (ELLIS, 2015). In this context, it has been reported that naturally infected cattle may shed the bacteria for about 40 weeks, while experimentally infected calves shed for up to 32 weeks (LEONARD et al., 1992). Independently of the use of host-adapted (e.g. Hardjo in ruminants), or incidental (e.g. Pomona in ruminants) serovars for experimental infections, renal colonization has been reported, in general 10-25 days after the infectious challenge (SLEIGHT et al, 1964; LITLE & SALT, 1976; RINEHART et al, 2012).

For a long time, it has been stated that urine leptospiral shedding to be intermittent (FAINE et al., 2000). It was first determined by the inconstant recovery of this microorganism by culturing (IN AD A et al., 1916). Nevertheless, it must be considered that culturing of leptospires presents serious limitations, mostly low sensitivity (CHIDEROLI et al., 2016), so failure on detection of leptospires cannot be neglected. Thus, it may be unclear if intermittence really occurs or if the failure on recovering leptospires from infected animals is a reflex of the low sensitivity of culturing. PCR has been widely used for the diagnosis of leptospirosis, with high sensibility and specificity (PICARDEAU, 2013; TAYLOR et al., 2015). It is a rapid and reliable method that may be used in large-scale. Additionally, frozen samples may be used for PCR without compromising the reaction (HAMOND et al., 2014). Considering the advances of PCR on the detection of carriers in leptospirosis, this study aimed to analyze the occurrence of intermittency on leptospiral shedding in naturally and experimentally infected animals.

MATERIALS AND METHODS

Study Design

In this study two experiments were conducted, both with the approval of the Ethics Committee of Universidade Federal Fluminense (UFF), Brazil (number 814/2016). The first one was conducted in naturally infected cows belonging to the Empresa Brasileira de Pesquisa Agropecuaria (Embrapa), Gado de Leite-Valenca. Rio de Janeiro, while the other was conducted on experimentally infected sheep in the Unidade de Pesquisa Experimental em Caprinos e Ovinos (UniPECO) on the school farm of the Universidade Federal Fluminense (UFF)-Cachoeiras de Macacu, Rio de Janeiro.

Experiment I

In the first experiment (naturally infected cows), 60 adult cows from a herd known to be endemically infected were studied. The animals remained altogether in the same pasture. Six urine samplings were made from each cow, at weekly intervals, totaling 360 samples. Urine was collected after intravenous furosemide administration, in sterile conical tubes (50ml) and then 1ml aliquots were transferred into microtubes, identified, conditioned at 4[degrees]C and sent to the laboratory.

Experiment II

The second experiment was conducted in three experimentally infected ewes. Each one received a single dose ([+ or -] [10.sup.8] leptospires) of different strains of Leptospira (strains Copenliageni Ll-130, Canicola LO-4 and Pomona Fro mm). Animals were infected by intraperitoneal route and were kept in separate pens. Animals were observed daily by veterinarians. Urine samples were collected as described in cows, but during eight times, at three days intervals, totaling 24 samples. Thus, the total number of samples (cows and ewes) was 384.

PCR

The DNA was extracted using the Promega Wizard SV Genomic DNA Purification System[R] (Promega, Madison, WI, USA). Primers were targeted to the lipL32 gene (regarded as present only in pathogenic leptospires), LipL32_45F-5'-AAG CAT TAC CGC TTG TGG TG-3' and LipL32_286R -5'-GAA CTC CCA TTT CAG CGA TT-3', which generate a fragment of 242pb (STODDARD et al., 2009). Briefly, primers were used in concentrations of 0.6|iM, 1.OU Taq polymerase, 2.4[micro]M MgC12, and 0.3m MdNTP in a final volume of 25[micro]L. One cycle of initial denaturation at 94[degrees]C for 2min, followed by 35 cycles of denaturation at 94[degrees]C for 30s, annealing primers to 53[degrees]C for 30s and 1min extension with 72[degrees]C and final extension cycle at 72[degrees]C for 5min. Strain L. interrogans serovar Copenliageni, Fiocruz L1-130 (ATCC BAA-1198) was used as a positive control.

RESULTS

Considering cattle, 26/60 (43.3%) animals tested negative in all samples and were considered as non-infected. For the remaining 34 (56.7%) cows that were PCR-positive at least once, only one (1.6%) presented positive in all samples, and seven (11.8%) were positive only in the last sampling, making impossible to evaluate the intermittency. Noteworthy, 26 of the naturally infected cows (43.3%) presented the typical intermittent pattern of urine leptospiral shedding (Table 1). In relation to the three experimentally infected sheep, all of them presented the typical intermittent pattern of urine leptospiral shedding, independently of the inoculated leptospiral strain (Table 1).

DISCUSSION

The results confirmed that when employing molecular tools there was intermittent shedding of leptospires, independently of the leptospiral strain, host species, natural or experimental infection. This shedding intermittency may have strong implications for the control of leptospirosis on livestock, mainly on the strategic decision about employing or not antimicrobial agents. Although, the whole-herd treatment approach has been recommended (MUGHINI-GRAS et al., 2014) it may be expensive and economically non-feasible. Besides the cost, other important aspects should be reminded, as the grace period of milk/meat after the usage of antibiotic therapy, as well as the enviromnental impact of the antibiotics usage. Therefore, employing antibiotic therapy cannot be performed indiscriminately and must be proceeded by a thorough identification of infected animals (MARTINS & LILENBAUM, 2017). Isolation of leptospires in tissues, urine and blood is considered the gold standard for the definitive diagnosis of leptospirosis (OIE, 2014). However, this isolation is usually achieved after weeks or months (VERMA et al., 2012), what makes this method not ideal for a rapid diagnosis (ADLER & DE LA PENA MOCTEZUMA, 2010). In contrast, PCR seems to be an alternative for a rapid and direct diagnosis of the infection. Nevertheless, this method is a poor indicator for the infecting serovar in a herd, and its implication for epidemiological studies is limited, expensive and difficult (ELLIS, 2015).

Results of our experiments clearly demonstrated that after negative results on PCR or culture of one single urine sample, animals cannot be reliably considered as non-infected, since infected animals may not be detected due to the intermittency. Considering the recent reduction of costs on molecular diagnostic methods, we consider that a careful serial analysis of urine samples for a more definitive and reliable individual diagnosis would be required for a successful control program of leptospirosis on a herd.

The low number of experimentally infected animals used in the presented study represents a limitation. Despite that, intermittence was clearly observed in all infected ewes. Other possible bias of this study was the possible interference of enviromnental conditions in naturally infected animals. Transmission of leptospirosis is influenced by enviromnental conditions, such as rainfalls (CORREIA et al., 2017), which may interfere in the exposure of those animals to leptospires, also influencing the reinfection and intermittency.

In conclusion this is the first study to describe the intermittent shedding of leptospires in urine by PCR. We suggest that control strategies may incorporate a serial analysis of urine samples for a more reliable individual diagnosis and treatment.

http://dx.doi.org/ 10.1590/0103-8478cr20170088.

Received 02.08.17

Approved 04.15.17

Returned by the author 06.05.17

CR-2017-0088.R1

ACKNOWLEDGEMENTS

The authors are thankful to Dr. Felipe Z. Brandao (UFF) and his research group for their help on the experiments. The authors are particularly thankful to Dr. Mario Balaro (UFF) for his permanent assistance, to Anahi Vieira (UFF, UFMS) who helped on PCR execution, as well as the staff of Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA). WL is Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) fellow. GM and BR are Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) fellows.

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

This study was approved by the Ethics Committee of Universidade Federal Fluminense (UFF), Brazil (number 814/2016).

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Bruno Ribeiro Rocha (1) Lorena Narduche (1) Clara Slade Oliveira (2) Gabriel Martins (1) Walter Lilenbaum (1) *

(1) Laboratorio de Bacteriologia Veterinaria, Departamento de Microbiologia e Parasitologia, Universidade Federal Fluminense (UFF), 24210-030,

Niteroi, RJ, Brasil. E-mail: whilenbaumMid.ulf.br. * Corresponding author.

(2) Empresa Brasileira de Pesquisa Agropecuaria (Embrapa), Gado de Leite, Valenca, RJ, Brasil.
Table 1--Status of naturally infected cows and experimentally infected
sheep for leptospiral urine detection after serial PCR results.

Status     Positive on     Negative on    Intermittent   Inconclusive
Species   all samplings   all samplings     shedding

Cows        1 (1.7%)       26 (43.3%)      26 (43.3%)      7(11.7%)
Sheep         0(0%)           0(0%)         3 (100%)        0(0%)

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Title Annotation:microbiologia; texto en ingles
Author:Rocha, Bruno Ribeiro; Narduche, Lorena; Oliveira, Clara Slade; Martins, Gabriel; Lilenbaum, Walter
Publication:Ciencia Rural
Date:Aug 1, 2017
Words:2392
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