INTRODUCTION

Brucella melitensis is the etiological agent of small ruminant brucellosis, although under particular epidemiological situations, goats and sheep may act as occasional hosts for other species of the genus Brucella (Rossetti et al., 2022). In addition, B. melitensis-infected flocks are the origin of cattle outbreak of brucellosis in mixed breeding systems (Muendo et al., 2012) and they are responsible for the highest number of human cases due to consumption of unpasteurized milk or milk products, or by direct contact with infected material (Blasco and Molina-Flores, 2011). In spite of being controlled in most first-world countries, the pathogen still produces an extensive negative impact on flocks in low- and middle-income nations, such as the Mediterranean region, the Middle East, Central Asia, Sub-Saharan Africa, and parts of Latin America, where goats and sheep are the major livestock species (Rossetti et al., 2017).

Abortion is the characteristic and perhaps the only noticeable clinical sign of brucellosis in goats and sheep (Rossetti et al., 2022), and vaccination is the best option to prevent and control this clinical manifestation (Blasco, 1997). The virulence-attenuated strain Rev.1, a B. melitensis mutant derived from a streptomycin-dependent variant of the virulent strain 6056 (Herzberg and Elberg, 1953), is the immunogen used worldwide against small ruminant brucellosis. However, due to the affinity that the Brucella species has for pregnant uteri, invasion of the genital tract, fetus colonization and abortion are some of the negative side effects retained by this vaccine strain (Alton, 1987; Jimenez de Bagues et al., 1989). To date, goats and sheep have been the preferred animal models to test the safety of the B. melitensis Rev.1 vaccine on pregnancy (Crowther et al., 1977; Jimenez de Bagues et al., 1989; Zundel et al., 1992; Higgins et al., 2017). Using primary hosts to test vaccine safety allows the direct evaluation of the effects under study, but these trials are costly, time-consuming and present significant challenges, including biosafety concerns and the requirement of valid biocontainment facilities, sometimes with undesirable performance. Because of these issues and ethical concerns associated with experimentation in large mammals, laboratory animal species serve as important tools for conducting brucellosis vaccine trials.

Mice (Mus musculus) are usually the experimental animal model of choice for evaluating the efficacy of the brucellosis vaccine (Darbandi et al., 2022; Grilló et al., 2012), due to their ease of handling, their cheapness (purchase, housing and food), their availability, and the existence of vast literature and specific laboratory protocol and reagents (Carvalho et al., 2016). However, they have not been extensively used to test the safety of brucellosis vaccines on pregnancy, including the Rev.1 strain (Elizalde-Bielsa et al., 2024). Guinea pigs are probably the most susceptible laboratory animal species to Brucella infection (Silva et al., 2011). They show a very high susceptibility to B. melitensis (Braude, 1951; Garcia-Carrillo, 1990), and like the natural hosts, virulent B. melitensis causes abortions when inoculated into pregnant females (Hensel et al., 2020). Guinea pigs have demonstrated to be an excellent model for testing the immunogenicity and efficacy of the Rev.1 anti-B. melitensis vaccine (McCamish and Elberg, 1962; García-Carrillo, 1986; Nicola et al., 2014), but the pregnant guinea pig model has not been explored to address the effects of this vaccine strain on pregnancy. The goal of this study was to asses the effects of subcutaneous (SC) inoculation of the vaccine strain B. melitensis Rev.1 on pregnant guinea pigs to evaluate the possibility of establishing a suitable laboratory animal model, in order to test and compare the safety on pregnancy of vaccine candidates against small ruminant brucellosis before testing them on natural hosts.

MATERIALS AND METHODS

Bacterial strains, media and culture conditions

The B. melitensis Rev.1 strain was obtained from the commercial vaccine (OCUREV®, CZ Vaccines, Pontevedra, Spain), kindly provided by SENASA (Official Veterinary Service of Argentina). The lyophilized strain was reconstituted according to the manufacturer’s instructions and, immediately, 0.1 ml of the suspension was diluted 1:10 in sterile distilled water and cultured in a Brucella agar base (BAB, Laboratorio Britania, Buenos Aires, Argentina) plate. After four days of incubation at 37°C, the bacteria were harvested, washed twice in sterile distilled water, and re-suspended in a frozen solution (sterile trypticase soy broth –TSB– containing 15% glycerol). Subsequently, the stock culture was dispensed in 1.5 ml microtubes and stored at -80ºC until use.

When needed, one vial of the Rev.1 strain stock culture was thawed in water bath at 37ºC and cultured in TSB (Laboratorio Britania) at 37ºC under shaking condition (180 rpm) with loose lids. After 48 h, the culture was harvested and washed in sterile distilled water and re-suspended in sterile phosphate saline solution (PBS, pH 7.2).

The actual number of viable bacterial/ml was retrospectively obtained by serial dilution in PBS and plated onto BAB for quantitation (Castaño-Zubieta et al., 2021). The inoculum was adjusted by dilutions in PBS as appropriate.

Guinea pigs

American short-haired female guinea pigs (Cavia porcellus) bred at the INTA animal facilities, weighing between 550 to 650 g, were used. Animal welfare was determined by daily clinical observation of the guinea pigs, their appetite, environmental interaction, body temperature, respiratory rate and stool consistency. The animals received water ad libitum and were fed with concentrate and fresh vegetables (spinach, chard, carrots, kale). Barnyards were dry-cleaned three times per week. A week after their arrival, the females mated naturally by introducing one male to every three females in a cage for 17 days (one guinea pig estrus cycle).

At late gestation (>50 days), all the experimental female guinea pigs were euthanized by intramuscular application of 0.5 ml of xylazine (2%) (Richmond, Bs. As., Argentina), followed by intracardiac overdose of sodium pentobarbital (Euthanyle; Brouwer, Bs. As., Argentina). Necropsies were performed to collect the uteri for further assessment of bacterial colonization and microscopic observation of the tissues. Blood for serum was recovered from all the experimental animals before euthanasia. All the animal procedures were approved by the Institutional Animal Care and Use Committee (CICUAE) of CICVyA-INTA under approval number 01/2020.

In vivo inoculation

Experimental design

To determine the behavior of Rev.1 strain in pregnant guinea pigs, the animals from groups A, B and C (three pregnant females in each) were inoculated SC between the third and the fifth week of pregnancy (Hensel et al., 2020) with 0.5 ml of inoculum containing 10⁸, 10⁷ and 10⁶ CFU of B. melitensis Rev.1, respectively (table 1). The control animals (group D, n = 3) were simultaneously inoculated SC with 0.5 ml of PBS. The experimental animals were euthanized one week (+/- 5 days) before the estimated day of delivery to avoid bacterial dissemination to the environment. Samples for bacteriological detection and histological observation were collected at necropsy.

Serology

Five ml of blood for serum were collected from all the experimental animals by intra-cardiac puncture under sedation. Brucella-specific antibodies were determined by buffer plate antigen (BPA) and complement fixation (CF) tests, following international recommendations (Alton et al., 1988; OIE, 2018). Briefly, for BPA test (Rosenbusch, Bs. As., Argentina), positive or negative results were determined by the presence or absence of visible agglutination, respectively. A scale was developed to categorize the degree of agglutination as 1) +++ strong, 2) ++ mild, 3) + weak and 4) – no agglutination. CFT was performed in microtiter plates (U bottom) as previously described (Foster et al., 2022). A hemolytic reaction of 50% or less at a dilution of 1:4 was considered as the minimum seropositive threshold (i.e., ≥ 20IU/ml).

Bacteriology

At necropsy, samples were taken from the spleen, mammary gland fluid, chorioallantoic placenta, amniotic fluid, and from the spleen, liver, lung and stomach content of the fetuses. For processing, one gram of the mother’s spleen, the chorioallantoic placenta, and the liver of the fetus were used; the fetal spleen and the diaphragmatic lobe of the right fetal lung were placed in sterile bags and mechanically homogenized until complete tissue disaggregation. Then, 1 ml of sterile distilled water was added, mixed and placed in a 1.5 ml microtube. One hundred µl of the suspension were cultured on a Farrell’s media plate for CFU counting. Simultaneously, a culture swab imbibed with mammary gland content, amniotic liquid and fetal stomach fluid were cultured in Farrell’s media by dissemination. All plates were observed daily until colonies appeared, and were maintained at 37ºC for up to 10 days if there was no colony growth. The mothers’ spleens were weighed immediately after recovery and before sampling. The following scale was used for evaluating the number of CFU / g of tissue or culture swab: - (negative, no growth), + (1-30 CFU), ++ (31-300 CFU) and +++ (>300 CFU).

Morphological analysis

To assess the histologic changes associated with the inoculation of Rev.1 in pregnant guinea pigs, samples from the mothers’ spleens, uteri, mammary glands, chorioallantoic placentas, and from the fetuses’ left lungs, spleens and livers were taken at necropsy. The tissues were fixed in 10% neutral buffered formalin and were then routinely processed and embedded in paraffin, sectioned at 5 m, stained with hematoxylin and eosin (H&E), and examined with light microscopy.

Statistical analysis

The CFU (logarithmic transformed) and spleen weight data were subjected to two-way and one-way analysis of variance (ANOVA), respectively. The statistical analyses were performed using the GraphPad Prism 6.0 software (San Diego, CA, USA) and P values < 0.05 were considered significant.

RESULTS

Clinical findings and pregnancy outcome

No adverse side effects were observed after the inoculation of Rev.1 during the course of the study, such as changes in behavior, loss of body weight, or local inflammation in response to the injection in any of the concentrations inoculated. With the exception of a guinea pig inoculated with 10⁸ CFU of B. melitensis Rev.1 that aborted 21 days p.i., all the other pregnant guinea pigs were euthanized one week before the estimated day of delivery to avoid bacterial dissemination to the environment. The number of gestations was from 1 to 5, with a crown to rump length of 9 to 14 cm (table 1). The length and general features of the intra utero fetuses (same gestation) were uniform, which indicates that no in utero fetal death had occurred up to the time of euthanasia.

Serological responses in pregnant guinea pigs

The agglutination test (BPA) results showed that all the treated guinea pigs presented a strong agglutination reaction, except for one animal inoculated with 10⁶ CFU of Rev.1 that presented a mild agglutination reaction (table 2). When the serum samples were analyzed by CFT, the guinea pigs inoculated with 10⁸ (group A) and 10⁷ CFU (group B) of Rev.1 showed a level of 1,000 international units per ml of complement fixation (CFIU/ml). In group C (10⁶ CFU), 2 animals presented 31 CFIU and the other animal was negative (<20 CFUI/ml). The control animals were all negative to both serological tests (table 2). Taken together, these results would indicate that the lower the dose of the inoculum, the longer the time required for a strong humoral immune response.

Bacteriological and histopathological analysis of tissues from pregnant guinea pigs

The B. melitensis Rev.1 was isolated from the spleen of all the treated guinea pigs, in direct relationship between the concentration of the inoculum and the number of CFU isolated, i.e., the higher the inoculum concentration, the higher the number of CFU of Rev.1 isolated from spleens (group A > group B > group C) (table 3). As expected, no Brucella was isolated from the spleens of the control animals.

Weight gain and spleen enlargement were observed randomly in inoculated animals, and there was no significant difference among the average spleen weight in the inoculated groups, regardless of the inoculum concentration used (10⁸ = 1.92 g, 10⁷ = 2.09 g, and 10⁶ =2.08 g; P > 0.05; table 3). In contrast, the average spleen weight of the control group was 1.43 g, but the moderate differences with the average spleen weight of the inoculated groups were not significant (P > 0.05). Histological images of spleens from pregnant guinea pigs inoculated with Rev.1 four weeks p.i. were characterized by a mild to moderate inflammatory infiltrate in the red pulp of histiocytes and epithelioid macrophages with fewer lymphoplasmacytic cells and congestion (figure 1 A). Uninfected controls had no histologic lesions in spleens. The degrees of histological alterations in the spleen did not correlate with the inoculum concentration, spleen bacterial load (CFU count) or spleen weight (table 3). In addition to enlargement, the only grossly visible alteration in spleens was a superficial abscess in a single animal inoculated with 10⁶ CFU of Rev.1.

The Rev.1 strain was also isolated in three (one from group A –A2- and two from group C –C1 and C3-) out of 7 samples processed from the treated animals (two samples from the treated animals were not processed), in concentration of <30 CFU / culture swab (table 4). The samples from PBS-inoculated guinea pigs were negative. The low level of colonization of the mammary gland correlates with a mild interstitial infiltration of histiocytic and plasmatic cells, regardless of the concentration of the inoculum (figure 1 B). Neutrophils were observed in the lumen of the acinus in few samples (figure 1 B). The mammary gland samples from the control group presented no lesions.

These results showed that Rev.1 has the ability to colonize and develop histopathological changes in the spleen and mammary gland of pregnant guinea pigs.

Placenta invasion and vertical transmission

Rev.1 colonized chorioallantoic placentas after its inoculation in pregnant guinea pigs, although at different levels according to the inoculum concentration. Thus, all the placentas from pregnant guinea pigs inoculated with 10⁷ and 10⁸ CFU of Rev.1 were colonized. Contrary, only two out of three placentas of the guinea pigs inoculated with 10⁶ were colonized, although at lower concentrations (table 4). Concordantly, the B. melitensis Rev.1 was isolated from most of the amniotic fluid of those allantoic sacs in which Brucella was isolated from the placenta. No Rev.1 isolation was reported from placentas o amniotic fluid in PBS inoculated group (table 4).

The histopathological analysis of the pregnant uteri indicated that, with one exception, all of the guinea pigs inoculated with the B. melitensis Rev.1, regardless of the inoculum concentration received, presented mild endometritis and myometritis with the presence of inflammatory cells, mainly histiocytes and lymphocytes. The exception was a pregnant guinea pig inoculated with 10⁷ CFU of Rev.1 (B3) which had severe myometritis, characterized by intense neutrophils, lymphocytes and macrophages infiltration (figure 2 A). Regarding the placenta, no lesions were observed in any of the Rev.1 or PBS inoculated guinea pig, with the exception of the placenta of the guinea pig with severe endometritis and myometritis (B3), which presented focuses of coagulative necrosis and arterial thrombi (figure 2 B).

Vertical transmission is the ability of a pathogen to colonize the fetus from an infected mother via transplacental route. This study showed that the Rev.1 strain is able to cross the placenta and infect the fetuses, being the number of infected gestations directly related to the inoculum concentration (table 4). The B. melitensis Rev.1 was randomly isolated from the lung, liver, spleen and abomasum content of the fetuses from inoculated groups, but not from the tissue samples processed from control animals. No gross or histologic lesions were detected in any of the fetal tissues examined (lungs, livers and spleens) in any group, except for a mononuclear cells infiltration in the lung interstitium in a fetus whose mother was inoculated with 10⁶ CFU of B. melitensis Rev.1 (figure 3).

DISCUSSION

In this study, we demonstrated that Rev.1 inoculated SC in guinea pigs at midterm gestation induces a humoral immune response, while invading and colonizing the placenta, mammary gland and fetuses via vertical transmission, which is similar to that observed in pregnant natural hosts inoculated with this vaccine (Jimenez de Bagues et al., 1989; Higgins et al., 2017). The susceptibility of pregnant guinea pigs to Brucella strains was initially demonstrated for B. abortus infection after parenteral inoculation (Bosseray and Diaz, 1974; Samartino et al., 1994) and, more recently, to B. melitensis via intratracheal challenge (Hensel et al., 2020). Likewise, the abortifacient capacity of B. abortus vaccine strains such as S19 and RB51 (Thomas et al., 1981; Yazdi et al., 2009) was also observed in 33% of the pregnant guinea pigs after being inoculated SC with 1x10¹¹ CFU of each strain (Samartino et al., 1996a). This would be the first time that the safety of Rev.1 has been evaluated in pregnant guinea pigs. Abortion, the main adverse effect after Rev.1 immunization, was not observed in this trial, with the exception of one animal, most probably because the assay was finalized early to avoid environmental dissemination of Brucella.

In control and eradication programs of brucellosis in small ruminants, the Rev.1 is inoculated by conjunctival instillation to a concentration of 1-2x10⁹ CFU. Ocular instillation not only confers similar immune protection than that induced by subcutaneous vaccination, but also reduces some of the disadvantages that occur when the vaccine is administered subcutaneously (Blasco and Molina-Flores, 2011). In any case, in this trial, the inoculum was administered SC because it is the preferable route for practical purposes and favors the comparison with other vaccines against brucellosis since it is the route initially chosen when novel vaccine developments are tested (Carvalho et al., 2016). Regarding the concentration of the inoculum, three different doses were tested. The doses chosen to evaluate the clinical and pathological consequences in guinea pigs pregnancy were 10⁸, 10⁷ and 10⁶ CFU of B. melitensis Rev.1, which are 1/10, 1/100 and 1/1,000 of that used in vaccination campaigns against brucellosis in small ruminants, respectively. It is broadly known that the official dose of the vaccine generates an abortion outbreak when goats and ewes are immunized between the second and fourth month of gestation (Jimenez de Bagues et al., 1989; Blasco, 1997). Thus the 10⁷ CFU dose maintains the CFU vaccine / live weight ratio between the target population of the vaccine and the laboratory animal species tested (10⁹ CFU / 60 to 80 kg -small ruminants- vs. 10⁷ CFU / 0.6-0.8 kg ―guinea pigs―). In addition, 10⁶ and 10⁷ CFU of Rev.1 have showed to be protective against challenge with virulent Brucella in guinea pigs without adverse effects (García-Carrillo, 1986; Nicola et al., 2014), and 10⁶ has been indicated as a recommended dose for Rev.1 vaccine potency test assays in guinea pigs (Alton, 1990). A dose higher than those used in this study (10⁹ CFU) had been similar to that used to vaccinate small ruminants and, consequently, it would have colonized the genital tract and generated an abortion outbreak in the inoculated guinea pigs. Conversely, a lower initial concentration of Rev.1 (≤ 10⁵ CFU) may not have enough time to reach a critical number to infect and colonize the genital tract (Alton, 1969).

Contrary to what have been reported by McCamish and Elberg (1962), no clinical evidence of adverse vaccine reaction was observed in guinea pigs following subcutaneous immunization with Rev.1, including local inflammation at the site of inoculation, loss of appetite or lethargy. The length and general characteristics of fetuses from the same gestation were not affected either. The differences in the length of the fetuses between different pregnancies could be due to the different gestational ages at the time of euthanasia, since the estrus was not synchronized and, therefore, some females became pregnant earlier than others, or by the number of fetus/pregnancy since, the greater the number of fetus, the smaller the size of each fetus.

The humoral immune response elicited by Rev.1 in pregnant guinea pigs 4 weeks p.i. was found to be consistent with that observed in the same animal species as well as in natural hosts by other reports. In those publications, the highest level of humoral immune response was reached between 14 and 45 days p.i. (McCamish and Elberg, 1962; Alton, 1969; Fensterbank et al., 1985; Castaño-Zubieta et al., 2021) even with lower doses of Rev.1. In the study reported here, the animals immunized with the lowest dose (group C; 10⁶) had the lowest humoral immune response, with a medium to high agglutinating response, and low or no complement activation activity. The low level of anti-Brucella specific antibodies in animals inoculated with a lower inoculum concentration was most probably due to the delay in the initial immune response that did not occur until the bacteria had reached a critical number at the site of infection necessary to spread.

After bacteremia, Brucella remains in cells of the reticuloendothelial system (RES) and induces splenomegaly in human and laboratory animal models due to an inflammatory response (Grilló et al., 2012). Based on that, many authors suggest that spleen enlargement and weight increase is a good indicator for brucellosis infection and it may replace bacteriology in experimental animal models (Braude and Spink, 1951; Thornton and Muskett, 1972; Garcia-Carrillo and Trenchi, 1974; Nicola et al., 2014). In this study, weight gain and spleen enlargement were randomly observed in the inoculated animals (table 3), and there was no significant difference between the mean spleen weights in the inoculated and control groups. Previous publications reported that the basal size and weight of the guinea pigs’ spleens show great variability (Garcia-Carrillo, 1977) and vary in animals inoculated with Rev.1 (McCamish and Elberg, 1962; Alton et al., 1988) or virulent strains (Braude, 1951). Accordingly, but difficult to explain, in this study, a large dispersion was observed in the weight of the spleens of two animals inoculated with 10⁷ CFU of Rev.1 (group B) in comparison with the weight of the spleens of the other inoculated animals (table 3). Interestingly, these two spleens were lighter than the spleens of the control animals (group D), even when the Rev.1 was re-isolated and the histological lesions were observed. Histological images of all the spleens of the guinea pigs inoculated with Rev.1 were characterized by a mild to moderate inflammatory cells infiltration of the red pulp, which was comparable to images informed by others in similar circumstances (McCamish and Elberg, 1962), but less severe than the histologic appearance of the spleens infected with virulent B. melitensis strains (Braude, 1951; Hensel et al., 2019).

It is well documented that the attraction of Brucella to placentas increases during the late stage of gestation (Samartino and Enright, 1996b), which is consistent with the abortions in the last trimester of pregnancy. However, placental colonization and vertical transmission are not always linked to abortion (Tobias et al., 1993; Hensel et al., 2020); that is more related to the moment of pregnancy, the inoculum concentration and the passage of time after inoculation. In goats and sheep, Rev.1 induces abortion 40 to 60 days after vaccination and when it is inoculated between the second and fourth month of gestation (Blasco, 1997). The trophoblasts are probably not mature enough to allow intracellular growth of Rev.1 during the first month of pregnancy; and, when inoculated in the last month, the strain does not have sufficient time to colonize the placenta and induce abortion. In this study, pregnant guinea pigs were inoculated at mid-term gestation (20-35 days of pregnancy), a period in which the placenta of this animal species had previously been shown to be permissive of colonization and, therefore, susceptible to abortion by brucellae (Samartino et al., 1996a; Hensel et al., 2020). Even when no abortions were observed, except in one experimental animal inoculated with 10⁸ CFU of Rev.1 (group A), the strain colonized the chorioallantoic placenta and was able to cross it. The lack of abortions, as well as the absence of lesions in the placenta and fetal organs, that are observed in goats and sheep immunized with Rev.1 (Jimenez de Bagues et al., 1989; Mazlan et al., 2021), can be mainly attributed to the experimental animals being euthanized 5 to 13 days before the estimated date of delivery. These differences in the gestational age may have affected the degree of susceptibility to Brucella infection and, consequently, the number of colonized fetal tissues or the isolated burden (table 4), as was also noted in other publications (Bosseray and Diaz, 1974). In addition, the lower level and number of colonized placentas and fetuses in animals inoculated with 10⁶ CFU of Rev.1 (group C) could be because, the lower the concentration of the inoculum, the longer it takes to establish an infection with clinical consequences (Alton, 1969).

Brucella spp. also has an affinity for udders It is eliminated through milk or colostrum, with consequences such as environmental contamination and potential risk of infection of dairy workers, consumers of unpasteurized dairy products and other susceptible animals residing in the same area, as well as vertical transmission to litters (Meador et al., 1989). Therefore, the potential shedding of the vaccine strain in milk and colostrum is an important parameter that needs to be evaluated with caution during Brucella vaccine developments. In this study, we demonstrated that Rev.1 can be present in colostrum and potentially be excreted. These data are in concordance with previous reports in which they isolated Rev.1 in milk or colostrum of small ruminants after being vaccinated during pregnancy (Jimenez de Bagues et al., 1989; Higgins et al., 2017). The low number of mammary glands in which Rev.1 was detected (3 out of 7 samples processed; 43%) may be due to the few samples tested (7 samples), the small sample size (one culture swab / sample), because Brucella spreads intermittently from each mammary gland (Morgan and McDiarmid, 1960), or all of the above. In any case, this study showed that B. melitensis Rev.1 is able to colonize the mammary glands of the guinea pig.

In conclusion, the mid-term pregnant guinea pigs inoculated subcutaneously with B. melitensis Rev.1 reproduced the undesirable side effects reported in pregnant small ruminants immunized with this vaccine strain, such as placental colonization, vertical transmission and affinity for udders. This model may be useful as a hypothesis-driven model to assess the safety of the modifications in Rev.1 formulation or dose concentration in pregnancy before testing them in natural hosts. Furthermore, it could eventually be extended to initially evaluate and compare the safety and vertical transmission of new brucellosis vaccine candidates in pregnant animals.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

Acknowledgments

ACKNOWLEDGMENTS

The authors would like to thank Mrs. Claudia Moreno and Mrs. Gladys Francinelli (IP-IPVET) for their technical assistance. This study was funded by INTA (Instituto Nacional de Tecnología Agropecuaria), Grant 2019-PD-E5-I102-001.

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