Serological diagnosis and molecular characterization of Leptospira spp. in the blood and urine of bovine females from refrigerated slaughterhouses

Diagnóstico sorológico e caracterização molecular de Leptospira spp. em sangue e urina de fêmeas bovinas de matadouros-frigoríficos

Leptospirosis is an important disease in the concept of One Health and it is caused by bacteria of the genus Leptospira, which cause economic and social losses involving domestic animals, wild animals, humans, and the environment (GROOMS, 2015; MONTE et al., 2015). Leptospirosis is an infectious disease that is considered occupational, since exposure to infected animals during work can infect slaughterhouse and rural workers, veterinarians, among others (GONÇALVES et al., 2006, 2013; SIMÕES et al., 2016). In the context of occupational disease, workers often lack knowledge about leptospirosis, resulting in failure to use, or misuse of, personal protective equipment (PPE). This makes the workers more susceptible to infection (CORRÊA et al., 2013).

Beef and dairy cattle breeding play a key role in the epidemiology of bovine leptospirosis, since bovine animals intermittently excrete Leptospira spp. in their urine, thus contaminating the environment and infecting humans and animals (LOUREIRO et al., 2013; SHAFIGHI et al., 2014). Similarly, leptospirosis persists in bovine herds through reproductive tract infections in both male and female animals. Thus microorganisms can be disseminated into the environment via post-abortion uterine discharge, fetuses, placenta, uterine infections, and infected semen (ELLIS, 1994).

After the 1980s, when antigens to detect the serovar Hardjo were included in the microscopic agglutination tests (MATs) of Brazilian laboratories, researchers were better able to understand the prevalence and subsequent epidemiology of this serovar in Brazilian herds. At this time, the serovar was already known in bovine species in Australia, Europe, North and South America (LILENBAUM, 1996; BOLIN; ALT, 1999; FAINE et al., 1999; CHIDEROLLI, 2016).

Epidemiological investigations of leptospirosis in bovine animals must take into account clinical signs, type of breeding management, and breeding site, among other variables. Nonetheless, serological diagnosis using a MAT to detect antibody, and molecular diagnosis to detect Leptospira DNA by PCR are also essential (FAINE et al., 1999; WHO, 2003; HAMOND et al., 2014; GROOMS, 2015).

According to the International Life Saving Federation of the World Health Organization (2003), the MAT is considered the gold standard method for the diagnosis of leptospirosis because it has high serovar and serogroup specificity. For this reason, it is the most common method used by researchers worldwide. On the other hand, different molecular techniques have been developed for DNA detection and subsequent identification of Leptospira spp. These are often used when it is necessary to work with fast, high-sensitivity, high-specificity methods (MAGAJEVSKI, 2002; ANZAI, 2006).

The advantage of these molecular methods is that they require minimal amounts of Leptospira DNA for amplification. Thus, different biological samples can be used, such as serum, cerebrospinal fluid, urine, feces, and tissues, and clinicians can obtain an early diagnosis of leptospirosis in both humans and animals. That is, the disease can be confirmed before antibody titers can even be detected, or when they are still low (BAL et al., 1994; ANZAI, 2006).

The northwest region has the largest bovine herd in the state of Paraná, with 2,084,593 animals (IBGE, 2015). Despite this, the data from this region are scarce regarding bovine leptospirosis, its relationship with public health, and its molecular characteristics. Thus, the objective of the present study was to detect Leptospira spp. antibodies and DNA in bovine females from two refrigerated slaughterhouses in the microregion of Umuarama, Paraná, Brazil.

The urine samples were subjected to molecular tests to detect Leptospira spp. DNA at the Molecular Biology Laboratory of the Animal Science Postgraduate Program, with emphasis on Bioactive Products, UNIPAR, and at the Laboratory of Leptospirosis, Universidade Estadual de Londrina (UEL).

To detect Leptospira spp. DNA samples were extracted immediately after each urine collection using the PureLink Genomic DNA Mini Kit (Invitrogen, USA). Subsequently, the DNA was subjected to nested PCR (n-PCR) using primers A (5’-GGCGGCGCGTCTITAAACATG-3’), B (5’-TTCCCCCCAT TGAGCAAGATT-3’), C (5’-CAAGTCAAGCGGAGTAGCAA-3’), and D (5’-CTTAACCTGCTGCCTCCCGTA-3’), as described by Mérien et al. (1992). The Platinum PCR SuperMix Kit (Invitrogen, USA) was used in all PCR reactions. The final product of the n-PCR amplification was subjected to electrophoresis in a 2% agarose gel containing ethidium bromide (0.05 µg/µL) and visualized using ultraviolet light in a transilluminator. The product’s molecular weight was estimated by comparison with a 100 bp molecular marker.

Samples considered positive after n-PCR were subjected to new DNA amplification using primers specific for the SecY gene: F (5′-ATGCCGATCATTTTTGCTTC-3′) and R (5′-CCGTCCCTTAATTTTAGACTTCTTC-3′) to identify and confirm the genetic species (AHMED et al., 2006).

Amplification and sequencing of the SecY gene PCR (AHMED et al., 2006) were performed for samples and the products of gene amplification were purified with a Purelink Genomic DNA extraction kit (Invitrogen Life Technologies, Eugene, OR, USA), quantified by a QubitTM Fluorometer (Invitrogen Life Technologies, Eugene, OR, USA), and sequenced on a ABI3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using forward and reverse primers. The contig were obtained by CAP3 and sequence quality was analyzed by visually in BioEdit software v. 7.2.5 (http://www.mbio.ncsu.edu/bioedit/). The nucleotide similarity was compared with all the sequences that were deposited in non-redundant database of GenBank using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The alignment was created in the BioEdit program using clustalW package and phylogenetic tree built using the MEGA 7.0.18 program (KUMAR et al., 2016).

The MAT yielded 22 (42.3%) serum samples considered reagent, of which 18 (81.8%) samples contained antibodies against a single serovar Wolffi (15 samples; 68.2%), Pomona (two samples; 9.1%), and Pyrogenes (one sample; 4.54%) with titers from 100 to 3,200. In four samples (18.2%), antibodies against two or more serovars were simultaneously detected, with titers from 100 to 400. It was not possible to characterize the most probable serovars in these samples (Table 1).

Table 1
Most probable serovars and titers detected by the MAT in 22 seroreagent bovine female samples from two refrigerated slaughterhouses in the microregion of Umuarama, Paraná, Brazil, 2016.

In the n-PCR, it was possible to amplify a 289 bp product in only one urine sample (1.9%). This single sample was then amplified again using primers for the SecY gene (549 bp). Importantly, this sample was considered negative in the MAT (Figure 1).  

BlastN software was used to compare partial sequences of the SecY gene, and the sample sequences were most similar to the genotype Hardjobovis of the species L. borgpetersenii, serovar Hardjo. The partial sequence of the SecY gene of sample 10 is deposited in the GenBank database under access number KY306666. In addition, the sample was grouped in the same phylogenetic cluster as Hardjobovis genotype of the serovar Hardjo and more phylogenetically distant from the genotype Hardjoprajitno (Figure 2).

Figure 1
Nested PCR for Leptospira spp. in bovine female urine samples from two refrigerated slaughterhouses in the microregion of Umuarama, Paraná, Brazil in 2016.

Legend: 2% agarose gel. Line 1: molecular marker (100 bp). Line 2: sample 10 with DNA amplification. Line 3: sample 11. Line 4: sample 12. Line 5: sample 13. Line 6: negative control. Line 7: positive control.

Figure 2
Phylogenetic tree of Leptospira spp. strains. The position of the sequence obtained from the bovine urine sample is highlighted, showing its relative position within the genus Leptospira. This representation was based on GenBank sequences from partial SecY genes. The “maximum likelihood” statistical method was used, and the reliability of the branches was validated through the generation of 1,000 bootstrap repetitions. Evolutionary analyses were performed using MEGA 7.0.18 software, and the sequence obtained in the SecY gene sequencing was deposited in the GenBank database under the access code KY306666.

Considering that the state of Paraná holds 9,314,908 bovine animals (IBGE, 2017), it is important that researchers ascertain whether leptospirosis is present in the region, and that they obtain information on circulating serovars and establish animal, human, and environmental health measures.

In the present study, 42.3% of the samples were reagent in the MAT, with titers ranging from 100 to 3,200. This confirms the presence of possible sources of acute and chronic infection in these animals (FAINE et al., 1999).

The disease persists and is disseminated within a population by infected animals or asymptomatic carriers that intermittently excrete the bacteria in their urine for extended periods. Therefore, to inform health prevention efforts and educational measures, it is important that researchers assess the prevalence of the infection in herds from refrigerated slaughterhouses. In this way, any possible occupational infections can be avoided (GONÇALVES et al., 2006; SIMÕES et al., 2016).

In the present study, antibodies against Wolffi (68.2%), Pomona (9.1%), and Pyrogenes (4.5%) were detected. The predominance of the antibody against the Wolffi serovar in this study corroborated the results of the state of São Paulo (SP) (LANGONI et al., 2000) and the state of Goiás (GO) (MARQUES et al., 2010). However, it conflicts with the research of the state of Maranhão (MA) (COELHO et al., 2014), the state of Rio de Janeiro (RJ) (HAMOND et al., 2014), and the USA (GROOMS, 2015), where the antibody against serovar Hardjo was detected with higher frequency. Thus, bovine animals are considered a maintenance host of the Hardjo serovar of Leptospira (FAINE et al., 1999).

There was no amplification of DNA in the urine samples that were considered reagents in the MAT, which suggests possible chronic infections, being only the establishment of the presence of antibody in the respective serum samples. However, in one of the non-reactive sera samples in MAT, it was possible to amplify DNA (549bp for the sec Y gene) in the urine, which characterizes that the respective animal was in the phase of leptospiruria, that is, eliminating live leptospiras by urine and may cause possible environmental contamination and make slaughterhouse workers susceptible to possible leptospirosis infection when not using or misusing personal protective equipment (FAINE et al., 1999; ANZAI, 2006).

Even though the antibody against the Hardjo serovar is less immunogenic in bovine species, DNA amplification showed that the respective animal was in the initial stages of infection. Therefore, the results of the present study show that it is important to combine serological and molecular diagnosis techniques (HASHIMOTO et al., 2017), because they complement one another. Thereby, even when no antibody was detected in the serum sample of one animal, there may be DNA in the urine sample of this animal. Thus, PCR is important in the early diagnosis of diseases, because it is sensitive in the initial stage and allows fast diagnosis, which in turn facilitates infection treatment and prevention (STODDARD, 2013; GALLOWAY; HOFFMASTER, 2015).

The sequencing of the SecY gene (VICTORIA et al., 2008) was one of a number of molecular techniques developed and used to characterize Leptospira isolates and biological samples known to be infected by Leptospira spp. In the present study, the sample was characterized molecularly as genotype Hardjobovis (Figure 2). This genotype belongs to the species L. borgpetersenii, which causes leptospirosis in production animals and is different from the genotype Hardjoprajitno since it has a smaller genome, and several genes related to survival in the environment, transport, and metabolite use have been lost. Consequently, researchers have concluded that these may be the reasons for the bacteria’s strict dependence on the host-host transmission cycle in bovine species (ELLIS, 1994; CHIARELI et al., 2012; KOIZUMI; YASUTOMI, 2012).

Chideroli et al. (2016), evaluated 15 urine samples from bovine females with a history of reproductive failures. The animals came from a dairy herd in Paraná and demonstrated the molecular characterization of the strain L. borgpetersenii serovar Hardjo Hardjobovis. Thus, our research corroborated with the previous study and confirmed the presence and circulation of this strain in the north and northwest region of the state of Paraná. We hope that the present study will stimulate new serological and molecular studies in these regions to characterize the epidemiology of this genotype, as well as its possible consequences in affected herds.

This was the first molecular confirmation of Hardjobovis-like L. borgpetersenii species in the urine of crossbred bovine females older than 36 months from slaughterhouses located in the microregion of Umuarama, Paraná. The results of this work show the presence of antibodies and DNA of Leptospira spp. in biological samples of bovine females from slaughterhouses is important to establish preventive measures related to One Health health avoiding economic losses related to herd and social related to workers and the environment.

We thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for the PROSUP scholarship and UNIPAR for research funding.