Leptospira spp. in Naturally Infected Dairy Cow from a Brazilian Border Region

Abstract

Leptospirosis is an important infectious disease, which can generate large economic losses, especially in the dairy herd. The pathogen that causes this disease may have its entry in Brazilian herds facilitated by the existence of a large extension of land borders. Therefore, the objective of this work was to investigate the presence of DNA and antibodies against Leptospira spp. in samples of vaginal mucus and serum from naturally infected bovine females from small rural dairy farms in a border region. Blood and vaginal mucus samples were collected from 70 Holstein cows, from small rural dairy farms between October 2017 and June 2018. The inclusion criteria for dairy cattle of any breed were aged over 2 years, not vaccinated against leptospirosis, and presenting a history of any reproductive problem such as abortion, stillbirth, repetition of heat, absence of heat, and lack of conception. Blood was collected by puncturing the coccygeal vein; for the collection of vaginal mucus, it was necessary to use a tampon with an applicator. For the detection of anti-Leptospira spp. antibodies, the sera were submitted to microscopic agglutination test (MAT) and, for DNA detection, the vaginal mucus was submitted to the PCR technique. Among the 70 cows, 42.86% had reagents in MAT and the most likely serovar was Wolffi (43.47%). In 74.28% of the vaginal mucus samples, it was possible to amplify the Leptospira spp. DNA. The results of this work show the presence of Leptospira spp. antibodies and DNA in samples of serum and vaginal mucus from naturally infected bovine females from small rural dairy farms in a border region (Brazil × Paraguay). These results demonstrate the importance of considering bovine females as potential vaginal carriers of Leptospira spp. Thus, it highlights the importance of further studies to better understanding of this issue, in addition to carrying out molecular and serological tests, to monitor the infection and further characterize epidemiological studies of leptospirosis in herds from regions that face this international frontier challenge.

Introduction

Brazil produced 35,169 tonnes of milk in 2019, and only in the first half of 2020, ∼18,660,153 L of milk were produced just in Brazil. From the total amount, 1,891,427 L came from the state of Paraná, which is considered the second-largest milk-producing state (FAO 2020, Instituto Brasileiro de Geografia e Estatistica—IBGE 2020) in Brazil. In this context, infectious diseases have a very important role related to the health of these herds, especially when they directly affect the production and reproduction of these animals. Among the different infectious diseases of the reproductive sphere, leptospirosis is highlighted, given the scope of reproductive symptoms and the possibility of reducing milk production (Cervante 2002, Da Silva Pinto et al. 2016, Fávero et al. 2017).

Leptospirosis is an infectious disease that affects animals and humans, caused by any pathogenic species of bacteria of the genus Leptospira spp. (Faine 1998), being one of the main diseases of importance for public health and for the agricultural sector (Brasil 1995, Chiareli et al. 2012).

Rodents are the main transmitters of this disease since they are resistant to disease and remain long-term carriers of infection. In addition, their urine and kidney tissue with alkaline pH are favorable for the survival of the bacteria, allowing colonization in the renal tubules and elimination of the bacteria via urine indefinitely. They are also identified as reservoirs of the serovar Icterohaemorrhagiae (Faine 1999). Although rodents are the main reservoirs of leptospirosis, other species of animals can also behave as maintenance hosts. This is the case of the bovine species that can harbor the serovar Hardjo, considered the most frequent in this animal species in Brazil (Faine 1999, Barcellos et al. 2003, Bharti et al. 2003, Hashimoto 2012, Hashimoto et al. 2017).

In bovine species, the spread and maintenance of the microorganism are caused mainly by the presence of infected animals or asymptomatic carriers, especially cattle, which eliminate the bacteria through urine, cervicovaginal discharges, aborted fetuses, and placenta (Faine 1999, Loureiro and Lilenbaum, 2020) and when clinical manifestations are present, they may be related to decreased reproductive performance, characterized by abortions, embryonic death, repetition of heat, stillbirths, the birth of weak calves, mastitis, and decreased milk production (Cervantes et al. 2002, Da Silva Pinto et al. 2016).

According to Brazilian law no. 6,634 of May 2, 1979, a border region is defined as being the region within a 150 km wide strip, parallel to the dividing line of the land boundary of the national territory (Brasil 1980). In this sense, Brazil has land borders with 10 countries. These borders are highly vulnerable due to the integration between these countries and the attempt to further economic expansion can directly or indirectly affect sanitary barriers, especially land-based ones (Souto et al. 2016, Pastre et al. 2020).

Due to the agricultural importance of dairy cattle in the western region of the Brazilian state of Paraná and also due to the proximity to the Republic of Paraguay, the objective of this work was to investigate the presence of DNA and anti-Leptospira spp. antibodies in samples of vaginal mucus and serum from naturally infected bovine females from small dairy farms located in a border region.

Materials and Methods

Ethical aspects

This project was approved by the Research Ethics Committee Involving Animal Experimentation (CEPEEA) of Universidade Paranaense (UNIPAR) under protocol 31974/2017.

Sampling and sample location

Blood and vaginal mucus samples were collected from Holstein cows from small rural dairy farms in the city of Guaíra, located in the western region of the state of Paraná, Brazil, from October 2017 to June 2018. This municipality is located on the border of Brazil with the city of Salto del Guairá in Paraguay (21 km) and for this reason, it is considered a border region.

For dairy cattle of any breed, the inclusion criteria were dairy cattle of any breed, older than two years of age, not vaccinated for leptospirosis, and those which had a history of any reproductive problem such as abortion, stillbirth, repetition of heat, absence of heat, and failure to conceive.

Blood and Vaginal Mucus Collection

Blood collection was performed by puncturing the coccygeal vein, from which ∼10 mL of blood was collected. In the laboratory, the serum samples were placed in sterile flasks free of RNases, DNases, and PCR inhibitors and kept at −20°C until the moment of the serological and molecular examinations.

For the collection of vaginal mucus from each cow, it was necessary to use a tampon with an applicator (Tampax^® Regular, Procter and Gamble, São Paulo, Brazil). A tampon was inserted into each cow's vagina for a period of 10 minutes and then inserted into a sterile tube with 20 mL of phosphate-buffered saline solution (Lilenbaum et al. 2008). The samples were placed in an isothermal box under refrigeration for subsequent molecular detection.

Serological detection

For the detection of anti-Leptospira spp. antibodies, the sera were subjected to the microscopic agglutination test (MAT), with live antigens (Faine 1999). Twenty reference serovars were used: Australis, Bratislava, Autumnalis, Butembo, Castellonis, Bataviae, Canicola, Cynopteri, Grippotyphosa, Hebdomadis, Copenhageni, Icterohaemorrhagiae, Panama, Pomona, Pyrogenes, Hardjo, Wolffi, Shermani, Sentot, and Tarassovi. The antigens were kept at 28°C for a period of 5 to 10 days in Difco™ Leptospira Enrichment (EMJH) medium (DIFCO^®, USA).

The sera that presented at least 50% of the agglutinated leptospires were considered reactive and the reactive samples were geometrically diluted in ratio two to determine the maximum positive dilution. A 1:100 dilution was used as the cutoff point (Myers 1985). The analysis of the results considered as the most probable serovar, the one with the highest titer (Vasconcellos et al. 1997), and the sera that presented coagglutination in the highest dilution were only considered reagents for Leptospira spp. (Almeida et al. 1994).

Molecular detection

For the detection of Leptospira spp. DNA, the vaginal mucus was subjected to molecular tests. DNA extraction from vaginal mucus samples was performed following the silica—guanidinium isothiocyanate technique (Alfieri et al. 2006). Subsequently, they were submitted to the PCR technique using the following primers A, 5′-GGCGGCGCGTCTITAAACATG-3′ and B, 5′-TTCCCCCCAT TGAGCAAGATT-3′ (Marianelli et al. 2007, Mérien et al. 1992). The final product of the PCR amplification was submitted to electrophoresis in 2% agarose gel containing ethidium bromide (0.05 μg/μL), and the 331 bp product was visualized in a transilluminator with ultraviolet light. This project was approved by the Research Ethics Committee Involving Animal Experimentation of Universidade Paranaense under protocol 31974/2017.

Results

In total, 43 small rural dairy farms were visited. However, 30 (69.77%) properties were discarded from this study because they had already vaccinated cows for leptospirosis in a period less than or equal to 8 months. In 13 (30.23%) properties, it was possible to carry out the project ,and it was possible to collect samples of blood and vaginal mucus from 70 animals.

Among the 13 properties chosen for this study, 7 (53.84%) showed reactive animals in the MAT, and in 11 (84.61%), it was possible to detect the Leptospira spp. DNA. However, only in seven (53.84%), both the presence of antibody and the presence of Leptospira spp. DNA were verified.

From the total of 70 cows, 30 (42.86%) were reactive in MAT with titers ranging from 100 to 1600, and in 7 (23.33%), it was not possible to identify the most likely serovar. The most likely antibodies against serovars were Wolffi in 10 (43.47%) samples, followed by Pomona in 4 (17.39%), Butembo in 3 (13.04%), Hardjo and Icterohaemorrhagiae in 2 (8.70%) each, and Grippotyphosa and Tarassovi in 1 (4.35%) each.

From the vaginal mucus samples, in 52 (74.28%), it was possible to amplify the Leptospira spp., and of these 27 (51.92%) were also reactive in the MAT.

The results of the reactive samples in the MAT (most likely servers) and in the PCR are presented in Table 1.

Table 1.

Most Likely Serovars and Titers Detected in the Microscopic Agglutination Test and PCR for Leptospirosis in Serum and Vaginal Mucus Samples from Holstein Cows from Small Rural Dairy Farms Located in a City in the Border Region (Brazil x Paraguay), 2017

Animal	MAT		PCR
Animal	Titration	Most likely serovar	PCR
Dairy farm 1
1	200	Hardjo	+
2	800	Wolffi	+
3	200	Wolffi	+
4	200	Wolffi	+
5	200	Wolffi	+
6	—		+
7	—		+
8	100	Ictero	+
9	800	Wolffi	+
Dairy farm 2
1		NI	+
2	400	Wolffi	+
3		NI	+
4	100	Hardjo	+
5	200	NI	+
6	200	NI	+
7	200	Wolffi	+
8	200	Pomona	+
Dairy farm 3
1	—		+
2	—		+
3	—		+
4	—		+
5	—		−
Dairy farm 4
1	—		+
2	—		+
3	—		+
4	—		+
5	—		−
Dairy farm 5
1	100	Pomona	+
2	—		+
3	—		−
Dairy farm 6
1	—		+
2	200	Butembo	+
3	400	Wolffi	+
4	—		+
Dairy farm 7
1	—		+
2	—		+
Dairy farm 8
1	200	Butembo	+
2	100	Butembo	+
3		NI	+
4	—		+
5	—		+
Dairy farm 9
1	—		−
2	—		−
Dairy farm 10
1	—		−
2	—		−
Dairy farm 11
1	—		−
2	—		−
3	—		−
4	—		−
5	—		−
Dairy farm 12
1	—		−
2	100	Pomona	+
3		NI	+
4	—		+
5	—		−
6	—		+
7	800	Wolffi	+
8	—		+
9	—		+
10	—		−
11		NI	−
12	—		−
13	100	Tarassovi	+
Dairy farm 13
1	—		+
2	400	Gripo	+
3	—		+
4	100	Ictero	+
5	1600	Wolffi	+
6	—		−
7	800	Pomona	+

MAT, microscopic agglutination test; NI, most likely serovar not identified.

Discussion

The presence of infectious diseases, more specifically leptospirosis, in dairy herds can cause an increase in the interval between calves, subfertility, decreased production, and quality of milk in bovine females. In addition, it can cause an increase in the disposal of animals which, as a consequence, affects the entire milk production chain (Wapenaar et al. 2017, Abdisa 2018, Moreira et al. 2019). Thus, it is essential to characterize the presence and epidemiology of leptospirosis in dairy herds to maintain the economic viability of production.

It is already known that one of the main routes of transmission of leptospirosis is through the urine of infected animals. However, recent studies have shown that there are important extrarenal reservoirs in cattle, pointing semen and vaginal fluid as new possibilities for a possible venereal transmission of this disease, but the investigation of these fluids is still little explored in epidemiological investigations (Loureiro et al. 2016, Pires et al. 2018).

From the 43 rural properties visited in this study, only in 13 (30.24%), it was possible to carry out the project, since the choice of nonvaccinated animals for leptospirosis was a selection criterion. This situation is due to the fact that, in the serological diagnosis, titers equal to 100, 200, or even 400, can be caused by a vaccine response (in animals already vaccinated) or even correspond to a previous infection or in an early stage of the disease. Therefore, these other properties were excluded from this study to avoid reactions considered false-positive (Santos et al. 2018).

The serological results (42.86%) of this work differ from the research by Hashimoto et al. (2015), also performed in the state of Paraná and using only naturally infected animals, where the serological prevalence was found in the regions: Center-South equal to 48.09%, Center-West and North with 47.29%, and the Northwest with 41.13%, while the western region of the state of Paraná had a prevalence of 22.96%. Related to serovar, in this study, there was a predominance of serovar Wolffi, also detected as more prevalent in the bovine species in other studies in Brazil and Paraguay (Szwako et al. 2015, Furquim et al. 2021). These serological results may have been influenced by the difference in the prevalence of animal leptospirosis in the respective locations, by the period in which the study was carried out, and even by the number of animals present in these regions, which may have provided greater or lesser probabilities of infection in bovine females (Faine 1999).

Still, the serological results of the present study show us that leptospirosis is present in dairy herds in the city of Guaíra (PR) since the rural properties of this study did not have a history of vaccination against leptospirosis. Therefore, this prevalence is characterized by natural exposure to Leptospira spp., which demonstrates the epidemiological picture of this disease during the study period (August 2017–April 2018).

In dairy cattle, this natural exposure to the respective etiological agent may occur due to the presence of carrier animals, the elimination of the bacteria in the urine, subsequently causing environmental contamination, and the high turnover of the animals to obtain cattle with more satisfactory milk production (Jumper 2021). Another relevant point is that the region of this study borders Paraguay and, consequently, there must be frequent transit of cattle from one country to another due to more attractive prices, which can increase the risk of leptospiric infection in both countries (Niang et al. 1994, Faine 1999, Pastre et al. 2020). Leptospira infection has also been reported across the border, in Paraguay, where Paraguayan researchers have already demonstrated the presence of antibodies anti-Leptospira spp. in cattle herds (dairy and beef) as the study by Szwako et al. (2015) that detected 45.78% of reactive animals also using the MAT as a diagnosis, and the authors concluded that the environmental conditions (temperature and humidity) were favorable for the survival of the microorganism and subsequent transmission of the disease in that location.

These results highlight the importance of land borders in terms of the possible transmission of infectious diseases from cattle herds when in transit from one country to another.

In this study, it was possible to amplify Leptospira spp. DNA (331 pb) in 74.28% of vaginal mucus samples. These results are in line with those of Di Azevedo et al. (2021), Loureiro et al. (2016), Loureiro et al. (2017), and Pires et al. (2018), who, using molecular tests, confirmed the presence of Leptospira spp. DNA in the follicular fluid, cervicovaginal mucus, in vaginal fluids, and in the uterus of the animals. Such findings highlight the need for a new look at this disease, where the collection of genital material and the use of molecular techniques to identify the DNA of this bacterium could be approaches to be considered in the control and prevention of leptospirosis in herds (Loureiro and Lilenbaum 2020).

The present work achieved positive results, both in serological tests and also in molecular tests, corroborating the results of Hashimoto et al. (2017), where the animals, even with a titration below 100, were positive in the PCR test. However, in this study, the animals' urine was used. In this sense, the presence of antibodies against Leptospira spp., also confirmed by molecular testing, both in the urine and in the vaginal mucus of the animals, exposes the importance of combining these two diagnostic tests (Hashimoto et al. 2017), mainly for the characterization of epidemiological studies of the reproductive sphere in herds.

Titrations ranging from 100 to 400 in the serum agglutination test in unvaccinated herds may show the presence of animals with early leptospirosis or animals with chronic infections (Picardeau 2013, Santos et al. 2018). In the present study, despite a low titer in the serological test, there were positive samples in the molecular test, showing the importance of serological monitoring added to the molecular one, since these can be considered complementary where serology can work as monitoring the herd, while the molecular test shows the presence of Leptospira spp., which is being eliminated in the environment (Hashimoto et al. 2017), being important for new infections in cattle, other animal species, and even humans.

In this work, it was found that 53.84% of the rural properties presented positive results in the serological test for the different serovars of Leptospira spp., as well as presented DNA in the molecular test using cervicovaginal mucus samples. These results demonstrate that the bovine females, in addition to presenting antibodies against Leptospira spp., harbored the bacteria in the cervicovaginal region, which suggests the possibility of venereal transmission (female × male) between animals, as evidenced by the studies by Loureiro et al. (2016).

Dewes et al. (2020), Oliveira et al. (2018), and Pastre et al. (2020), as well as the authors of the present study, found the presence of Leptospira spp. antibodies in their studies in the border region, and Oliveira et al. (2018) and Pastre et al. (2020) also obtained positive samples in molecular tests. These results clearly show the presence of leptospiral infection in dairy cattle and, with porous land borders and cross-border movement of cattle, this can affect livestock health and productivity and cause economic loss in both countries (Ungerer and Reiss 2017).

It was found that 69.76% of the small farms in this study have the habit of practicing vaccination for leptospirosis and this demonstrates the knowledge and commitment of rural producers regarding the importance of the health of the herd.

Conclusion

The results of this work show the presence of Leptospira spp. antibodies and DNA in samples of vaginal mucus and serum from naturally infected bovine females from small rural dairy farms in a border region (Brazil × Paraguay). These results demonstrate the importance of considering bovine females as potential vaginal carriers of Leptospira spp. Thus, it highlights the importance of further studies to better understanding of this issue, in addition to carrying out molecular and serological tests, to monitor the infection and further characterize epidemiological studies of leptospirosis in herds from regions that face this international frontier challenge.

Footnotes

Acknowledgments

The authors thank Virbac; the Coordination for the Improvement of Higher Education Personnel (CAPES); CNPq (process: 310529/2017-4 - CP 12/2017); and the Universidade Paranaense for the financial assistance granted to this research.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

The authors declare that there is no funding for this study.

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