Infectivity and virulence of leptospiral strains of serogroup Sejroe other than Hardjo on experimentally infected hamsters

Abstract

To assess the virulence of leptospires from the serogroup Sejroe (from ruminants), hamsters were tested against 12 strains. Three Guaricura strains induced severe lethal disease, in contrast to the Hardjo strains. Although with the preliminary outcomes, this finding may be useful for the control of bovine leptospirosis in the Americas, where Guaricura is prevalent.

Bovine leptospirosis is a disease of great economic importance as it can lead to infertility, estrus repetition, and abortions [1]. Cattle are common renal carriers of leptospires, shedding viable bacteria in the urine and thereby contaminating the environment. Although cattle may harbor and shed many leptospiral strains from different serogroups [2], it is well known that the major agents of bovine leptospirosis worldwide are members of the serogroup Sejroe [3, 4]. Among the Sejroe strains, Hardjo seems to be the most common serovar in the two genotypes Hardjoprajitno (Leptospira interrogans) and Hardjobovis (Leptospira borgpetersenii) [2, 5]. More recently, another serovar of the same serogroup, named Guaricura (Leptospira santarosai), has emerged as a major agent of bovine leptospirosis in the Americas [6].

The physiopathology of reproductive disease first needs to be elucidated. There are many gaps in the knowledge about the host-agent relationship, and studies on experimentally infected animals are limited due to the low virulence of Hardjo strains on biomodels. The most common biomodel for leptospirosis studies is the golden Syrian hamster [7], and Hardjo strains are usually not virulent to that animal [8]. This impairs a variety of studies and compromises a full understanding of the physiopathology of the infection.

In this context, little is known about the virulence of Sejroe strains other than Hardjo. Although apparently predominant, Hardjo strains are not the only ones to infect cattle, and to our knowledge, no other strain of the serogroup Sejroe has been tested for infectivity and virulence on hamsters. The aim of this study is to evaluate the infectivity and virulence on the hamster model of Leptospira strains from the serogroup Sejroe obtained from ruminants (mainly bovine) in Brazil.

Leptospira strains (n = 12) were isolated from cattle (10), sheep (1), and buffalo (1) from 1967 to 2016 in Brazil. The used strains were classified through the sequencing of rrs and secY genes and by serogrouping (MAT) using specific polyclonal antisera [6, 9]. The descriptions of the Variable Number Tandem Repeat (VNTR) patterns of the inoculated strains were characterized [10]. These belong to the Collection of Bacterial Cultures of Veterinary Interest (Universidade Federal Fluminense, Brazil, www.labv.uff.br). A virulent L. interrogans serovar Copenhageni strain Fiocruz L1-130, provided by Prof. Odir Dellagostin, Federal University of Pelotas in Rio Grande do Sul, Brazil, was used in all experiments as the reference pathogen [11]. All Leptospira strains were cultivated in liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (Difco Laboratories) at 29 °C and stored in liquid nitrogen until use. Nine strains presented a maximum of six passages in vitro before the first freezing and three strains (BovG, M04-98, 2012_OV5) presented an unknown number of subcultures, as described in Table 1.

Table 1.

Leptospiral strains from the Sejroe serogroup used in the experimental infection in hamsters

Strain	Source	Origin	Species	Serovar	Infectivity		Virulence	Reference
					Blood culture at 4th day	Renal tissue PCR*
L1-130**	Human	Urine	L. interrogans	Icterohaemorrhagiae		NT	+	11
2015_U349	Cattle	Urine	L. interrogans	Hardjo	−	+	−	2
2014_U140	Cattle	Urine	L. santarosai	Guaricura	−	+	−	6
2014_VF66	Cattle	Vaginal fluid	L. santarosai	Guaricura	+	NT	−	6
U214/2013	Cattle	Urine	L. santarosai	Guaricura	+	NT	−	12
2014_U81	Cattle	Urine	L. santarosai	Guaricura	+	NT	+	6
2014_VF237	Cattle	Vaginal fluid	L. santarosai	Guaricura	+	NT	+	9
2013_VF52	Cattle	Vaginal fluid	L. santarosai	Guaricura	+	NT	+	13
BovG	Cattle	Kidney	L. santarosai	Guaricura	−	−	−	14
M04-98	Buffalo	Urine	L. santarosai	Guaricura	−	−	−	15
2014_U76	Cattle	Urine	L. santarosai	Guaricura	−	−	−	6
2015_U376	Cattle	Urine	L. interrogans	Hardjo	−	−	−	2
2012_OV5	Ewe	Vaginal fluid	L. interrogans	Hardjo	−	−	−	16

^*Only conducted on strains that were not recovered from the blood; NT not tested

^**Reference strain, used as positive control

The trials were performed after approval from the animal ethics committee of the Universidade Federal Fluminense (611/2015). Female golden Syrian hamsters (Mesocricetus auratus) 6–8 weeks of age and 80–120 g in weight were purchased from breeding facilities, housed in ventilated racks, and submitted to an adaptation period of 72 h prior to the tests at a temperature of 20–22 °C, a light/dark photoperiod, access to feed and water ad libitum, and an enriched environment. All procedures occurred under a biosafety cabinet [17].

The strains were passed up to four times in hamsters [18, 19] with adaptations. One hamster was used per passage [20] for each strain, with a total of 52 animals. For this purpose, strains were thawed and cultured in liquid EMJH for 7 to 15 days, depending on the isolate. Each strain was inoculated in hamsters only once the culture had reached 10⁸ leptospires per milliliter concentration. Each hamster was inoculated intraperitoneally with 1.0 mL of leptospiral culture (10 ⁸ leptospires) using a sterile tuberculin syringe and a 23 3/4 in. gauge needle. Each animal was monitored daily for signs of infection. Four days after inoculation (day 4 post-infection [p.i.]), blood (0.3 mL) was collected by gingival puncture [20] for the recovery of that isolate in EMJH and for confirmation of the infection. Re-isolated strains were serogrouped to confirm their identity [6]. The virulence of the strains in hamsters was estimated qualitatively for up to 21 days p.i., evaluating the severity of pain based on the Mouse Grimace Scale (MGS) [21] and external clinical signs (presence of epistaxis, hematochezia, and hematuria). Euthanasia occurred once pain was considered severe with irreversible clinical signs of acute leptospirosis or on the 21st day p.i. regardless of the animal’s clinical condition, according to the Brazilian guidelines of the Council of Veterinary Medicine. For euthanasia, an overdose of anesthetics (100 mg/kg of 10% ketamine and 10 mg/kg of 2% xylazine) administered intraperitoneally was used [19]. When the animal was unconscious with a loss of pedal reflexes, cardiac puncture exsanguination was performed for endpoint and necropsy. At this moment, the kidneys were macerated and used for leptospires culturing for 4 weeks and then examined using dark-field microscopy.

The strains were assessed (i) by their infectivity, given by the recovery of the inoculation strain in the blood of the animal at day 4 p.i. or by PCR positivity of the renal tissue samples, and (ii) by their virulence, given by the ability to determine acute disease and eventually death in the animal inoculated up to 21 days p.i.

The results are presented in Table 1. Out of the 12 tested strains, seven (2013_VF52, 2014_VF237, 2014_VF66, 2014_U81, 2014_U140, U214/2013, and 2015_U349) were recovered by culturing from blood on day 4 p.i. or were detected by renal tissue PCR, and were considered infective to hamsters. The other five strains (two L. interrogans serovar Hardjo and three L. santarosai serovar Guaricura) were neither recovered by culture nor detected by PCR, and were thus considered non-infective and avirulent.

Among the seven infective strains, four did not lead to acute clinical disease. In contrast, three (2013_VF52, 2014_VF237, and 2014_U81) led to a lethal infection in the hamsters and were considered virulent. The 2013_VF52 strain was fatal since the first passage in vivo and killed the hamsters on day 4 p.i. in all four passages. This strain presented severe clinical signs (ataxia, back arching, tachypnea, and deepening of the abdominal region), and on the fourth passage, hemorrhage was detected. Necropsy findings include pulmonary petechiae, mild jaundice, hyperemia and enlargement of uterus, and blackened bile and urine.

The virulence of the 2014_VF237 strains increased throughout the passages, killing the hamster only at the fourth passage, presenting similar clinical signs as 2013_VF52, although visible hemorrhage did not occur. The necropsy findings included hemorrhage in the lungs, kidney, and intestines and a friable spleen.

The most interesting outcomes of the present study were the important differences in infectivity and virulence observed among the strains, particularly for those belonged to the same species (L. santarosai) and serogroup (Sejroe). Regarding the genotypic characterization, it has been recently reported (Jaeger et al., 2019) that those studied L. santarosai strains fit to the same clonal complex, despite the high intraspecific variability observed by MLST. It might be related to the diversity observed also in infectivity and virulence, despite the lack of information enrolled specifically to virulence-associated genes in those strains. These findings reinforce that experimental infections on animal models are mandatory for the determination of virulence of strains.

Hardjo strains are known to be usually not lethal, determining a chronic infection [22], and that finding was confirmed in the present study. In contrast, strains of the serovar Guaricura determined both chronic and lethal infections. Golden Syrian hamsters have been reported as good models for acute lethal infection by several Leptospira serovars. The high virulence of three Guaricura strains is a remarkable result, and lesions on hamsters could be comparable with exemplary virulent strains of L. interrogans, such as Icterohaemorrhagiae strain L1-130 [23] or Canicola strain LO4 [24]. It is remarkable that despite that difference of the infectivity and virulence among studied strains in hamster model, all original hosts of the virulent strains were cows apparently asymptomatic. In this context, we suggest that the virulence degree observed in hamsters is not related to the disease or renal carrier status in the original host.

Although with the preliminary outcomes and small sampling, the results were consistent during the in vivo passages. Thus, when an alteration of the virulence profile was observed, it was always progressive (increase of the virulence). In this context, we performed a virulence test with significantly reduced animal numbers, considering the 3Rs (replacement, reduction, and refinement). Infectivity and virulence were evaluated from the same animal, when bacterium was recovered from the blood on the 4th day p.i., by evaluation of clinical symptoms up to 21 days of infection, and after euthanasia by renal tissue cultures and urine. In contrast, previous studies have reported [18, 19] the usage of two hamsters per passage during four passages, totaling eight hamsters per strain, twice the number of animals used in our study.

In conclusion, it was observed that leptospiral strains of serogroup Sejroe other than Hardjo, particularly strains of the serovar Guaricura, may be infective and virulent to the hamster model, determining both chronic and lethal infections. This finding may be useful for experimental studies using hamsters as biomodels and increases the knowledge on the physiopathology of infection by strains of the serogroup Sejroe on livestock.

Author’s contributions

CB was involved in the original methodological design of the study. GM was involved in the supervision of the project. CB and GM led the writing of the research article. WL was in charge of supervising the study, and read and approved the manuscript’s final version.