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A pregnant mouse model for the vertical transmission of Brucella melitensis
The Veterinary Journal 200 (2014) 116–121
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A pregnant mouse model for the vertical transmission of Brucella melitensis Z. Wang a, S.S. Wang b, G.L. Wang c, T.L. Wu a, Y.L. Lv a, Q.M. Wu a,⇑ a Key Laboratory of Animal Epidemiology and Zoonosis of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Yuanmingyuan Xilu No. 2, Haidian District, Beijing 100193, China b College of Biological Sciences, Anyang Institute of Technology, Anyang 455000, China c College of Veterinary Medicine, Qinghai University, Xining 810000, China
a r t i c l e
i n f o
Article history: Accepted 19 December 2013
Keywords: Brucellosis Bacteriology Histology Pregnant mouse Vertical transmission
a b s t r a c t Abortion is the major clinical sign of brucellosis in animals but little is known about the underlying mechanisms. This study was designed to evaluate a pregnant mouse model for the vertical transmission of Brucella melitensis using four infectious doses: 103 colony-forming units (CFU), 104 CFU, 105 CFU, and 106 CFU. During the experimental period, no instances of abortion were recorded, but stillbirths were observed in the groups infected with doses of 104 CFU and higher. Regardless of whether the fetuses were stillborn or alive, transmission of bacteria to the fetus and bacterial replication in the cytoplasm of placental trophoblast giant cells were detected. A higher degree of bacterial colonization was found in the placenta than in the spleen or fetus. Doses of 105 CFU of B. melitensis or higher produced a severe, necrotizing placentitis similar to the pathological damage observed in ruminants. The data suggest that experimental murine brucellosis resembles ruminant brucellosis and represents a potential model for studying the pathogenic mechanisms of B. melitensis. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
Introduction Brucella spp. are facultative, intracellular, Gram-negative bacteria with a marked affinity for the reproductive tract of pregnant ruminants. It is important to understand the pathogenesis of Brucella infection in pregnant females because of its significant economic impact on ruminant production. Vaginal secretions and aborted fetuses are the most important sources for infection of other animals and also represent an occupational source of infection for humans (Thoen et al., 1993; Neta et al., 2010). During acute brucellosis in pregnant cows, up to 85% of the bacteria are found in cotyledons, placental membranes, and allantoic fluid (Anderson et al., 1986b), resulting in placentitis, fetal death, and abortion (Samartino and Enright, 1993; Tobias et al., 1993). Although the mechanisms underlying placental localization, trophoblast tropism, and abortion associated with ruminant brucellosis are poorly understood, localization of bacteria in chorioallantoic trophoblasts has been shown to be important in the pathogenesis of many reproductive diseases of ruminants. For example, Coxiella burnetii and Chlamydia psittaci have been observed to fill the chorioallantoic trophoblasts of aborted placentas in sheep and goats (Jubb et al., 1985), and Listeria monocytogenes ⇑ Corresponding author. Tel.: +86 10 6273 3901. E-mail address: [email protected] (Q.M. Wu).
initially infects placental villous syncytiotrophoblast (Lecuit et al., 2004). Once Brucella is internalized by the erythrophagocytic trophoblastic epithelial cells from the maternal circulation (Anderson et al., 1986a), the internalized bacteria replicate within the rough endoplasmic reticulum, resulting in secondary infection of adjacent trophoblastic epithelial cells (Anderson et al., 1986a; Meador and Deyoe, 1989). The placenta is known to play an integral role in the pathogenesis of congenital infections and serves as an important barrier against infection. The structure of the bovine placenta is different from that of the murine placenta, but an infectious abortion model in pregnant mice could serve as a useful tool for investigating the mechanisms underlying Brucella pathogenesis. Several studies have addressed the induction of abortion associated with B. abortus infection in pregnant mice (Tobias et al., 1992, 1993; Kim et al., 2005; Watanabe et al., 2008) and have demonstrated transmission of bacteria to the fetus, bacterial replication in the placenta, and high abortion rates (Kim et al., 2005). Although B. abortus is the most important pathogen that induces abortion in pregnant cattle, infection with B. melitensis can also frequently cause placentitis or abortion (Anderson et al., 1986a, 1986b; Meador and Deyoe, 1989; Samadi et al., 2010). In 2007, we isolated a B. melitensis strain from an aborted bovine fetus in the Inner Mongolia region of China, which was designated B. melitensis NI (Liu et al., 2012). Considering the many differences
1090-0233/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2013.12.021
Z. Wang et al. / The Veterinary Journal 200 (2014) 116–121
between B. abortus and B. melitensis, including their biological characteristics, antigenicity, and host selectivity, in addition to the lack of studies addressing the induction of abortion due to B. melitensis infection in pregnant mice, we used in the present study B. melitensis NI to infect pregnant mice. Combined bacteriological and histopathological examinations were performed following the administration of various infective doses to evaluate B. melitensis infection in pregnant ICR mice and to establish an experimental mouse model for the vertical transmission of B. melitensis. Materials and methods Ethics statement All animal research was approved by the Beijing Association for Science and Technology (approval: SYXK, Beijing 2007-0001), and the animal research complied with the Beijing guidelines for laboratory animal welfare and ethics of the Beijing Administration Committee of Laboratory Animals. Bacterial strain and media B. melitensis NI is an epidemic strain that was previously isolated from an aborted bovine fetus from Inner Mongolia in our laboratory (liu et al., 2012). This strain, which is also referred to as the smooth virulent B. melitensis strain biovar 3, induces abortion in pregnant cattle, sheep, and goats. The complete NI genome was sequenced in our laboratory (GenBank accession numbers CP002931 and CP002932). This Brucella strain was routinely grown in tryptic soy broth (TSB) or tryptic soy agar (TSA) at 37 °C. All work with the virulent Brucella strain was performed in biosafety level 3 facilities at China Agricultural University.
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Statistical analysis Student’s t test was applied to analyze the bacteriology data. P < 0.05 was considered significant.
Results B. melitensis NI infection causes stillbirth in pregnant mice at a low infectious dose All 20 mice remained clinically normal for the duration of the experiments based on visual assessment, and no abortion was observed. However, at necropsy, stillbirth, characterized by a lack of heartbeat, or small fetal size and resorbed fetuses, which were malformed or autolyzed, was observed (Fig. 1). The fetuses recorded as resorbed had previously implanted in the uterine wall but were so damaged that they displayed no remaining recognizable fetal structure compared to other fetal units at the same stage of gestation. As shown in Table 1 and Fig. 1, the four mice infected with 106 CFU of B. melitensis NI displayed an average of one or two stillbirths or resorbed fetuses. Following infection with 105 CFU or 104 CFU, 50% of the mice (2/4) exhibited stillbirths. No stillbirths were observed in the group infected with 103 CFU or the control group. However, even in the 106 CFU infection group, one mouse showed a stillbirth rate that was not greater than 30%. These results indicated that B. melitensis NI was pathogenic for pregnant mice, even at a low infectious dose of 104 CFU/mouse.
Mice Female 6- to 10-week-old ICR mice were individually mated with male 6- to 10week-old ICR mice. The day on which a vaginal plug was observed was recorded as day 0.5 of gestation. The normal gestational time for these mice is 19 days. The mice were all purchased from the Weitong Lihua Laboratory Animal Services Centre (Beijing, China), bred in individually ventilated cage rack systems, and subsequently transferred to the biosafety level 3 facilities of China Agricultural University at the beginning of the experiments. All experiments involving animals followed the regulations enacted by the Beijing Administration Office for Laboratory Animals. Experimental infection To study bacterial colonization and the development of placental lesions and abortion, groups of four pregnant mice each were infected intraperitoneally (IP) with 103, 104, 105, or 106 colony-forming units (CFU) of B. melitensis NI in 0.1 mL of phosphate buffered saline (PBS) on day 4.5 of gestation. The exact injection doses expressed in CFU were verified retrospectively via serial dilution, plating, and counting. Four additional mice received 0.1 mL of PBS as a control. On day 18.5 of gestation, all mice were euthanased for bacteriological and histological analyses. Fetal development was evaluated and the fetuses were determined to be alive if there was a heart beat or dead if there was no heart beat (Kim et al., 2005). The minimal infectious dose that caused the proliferation of large numbers of bacteria in placentas and fetuses and induced stillbirth or abortion was determined. Bacteriology The placentas and the corresponding fetuses and spleens of each mouse were removed, weighed, and homogenized in PBS. The tissue homogenates were then serially diluted in PBS, plated on TSA plates, and incubated for 3–5 days at 37 °C under 5% (vol/vol) CO2, and the plates were monitored daily for growth. The bacteria recovered from each organ were counted. B. melitensis colonies were identified based on colony morphology, growth characteristics, and polymerase chain reaction (PCR) analysis using specific primers for B. melitensis as previously described (Huber et al., 2009). Histology The placentas from the mice that were not used for bacterial culture were fixed in 10% neutral buffered formalin and processed for routine histological examination with hematoxylin and eosin (HE) staining. To allow sensitive localization of B. melitensis bacterial colonization in the placental sections, immunohistochemical staining was performed using goat anti-B. melitensis serum as previously reported (Pérez et al., 1998). Non-infected murine placental sections were used as negative controls.
B. melitensis NI predominantly proliferates in the placenta To investigate bacterial multiplication in the infected pregnant mice, we examined the colonization of the B. melitensis NI strain in the spleens, placentas, and corresponding fetuses of the mice. The living or stillborn fetuses and the corresponding placentas of each mouse were analyzed. As shown in Table 1, numerous bacteria were isolated from the placentas of both living and stillborn fetuses in all mice, and the difference between the bacterial counts recorded in the placentas of living and stillborn fetuses was not significant (Table 1, P = 0.5127). In contrast, the bacterial load in the spleen was 2- to 3-log CFU/g lower than that in the placenta of the mice at all tested doses (Fig. 2A, P = 0.004336). Interestingly, both living and dead fetuses were found to be infected with B. melitensis NI, and the bacterial load was not significantly different between the living and dead fetuses (Fig. 2B, P = 0.8273). However, the number of bacteria recovered from the placentas, spleens, and fetuses in each group was positively correlated with the infectious dose (Fig. 2A and B). Immunostaining of bacterially infected placental specimens from each infection group revealed that B. melitensis was present in numerous trophoblast giant cells (TGCs), especially in the placentas of mice infected with 105 or 106 CFU, and the cytoplasm of the bacterially infected trophoblasts contained large numbers of B. melitensis surrounding cell nuclei. Furthermore, we observed intact TGCs exhibiting brown staining within the cytoplasm, indicating the presence of intracellular Brucella (Fig. 3). Although bacteria were also detected in the cytoplasm of fetal Kupffer cells (Fig. 4), the positive staining signal was much weaker than that observed in the placenta. Regardless of the location, the positive staining signal for Brucella was dose dependent. B. melitensis NI induces severe placentitis in pregnant mice Among the Brucella-infected mice, gross placental lesions were observed in mice receiving doses of 104 CFU or higher. The placentas of these mice varied in size and color. Those associated with
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Z. Wang et al. / The Veterinary Journal 200 (2014) 116–121
Fig. 1. Brucella melitensis NI infection caused fetal resorption and stillbirth in pregnant mice. Resorbed fetuses were characterized as malformed (A) or autolyzed (B); stillbirths were characterized by retarded growth (C), the lack of a heart beat, or small fetus size (D). Normal fetus (E).
Table 1 Bacteriological results for Brucella melitensis NI-infected pregnant mice. Dose/ mouse
Mouse Number
Fetus
Number
a
Placenta
Living Weight (g)a
Dead
Living
Bacterial number (log)a
Number
Weight (g)a
Bacterial number (log)a
Weight (g)a
Dead
Bacterial number (log)a
Weight (g)a
Bacterial number (log)a
103
1 2 3 4
12 14 17 16
1.16 1.04 0.91 0.92
(±0.04) (±0.05) (±0.04) (±0.03)
2.47 (±0.28) 2.91 (±0.22) 2.89 (±0.10) 2.77(±0.37)
0 0 0 0
/ / / /
/ / / /
0.14 (±0.01) 0.13 (±0.02) 0.14 (±0.01) 0.11 (±0.02)
5.63 5.82 6.04 6.49
(±0.56) (±0.74) (±0.65) (±0.74)
/ / / /
/ / / /
104
1 2 3 4
15 14 13 13
0.92 0.97 0.98 1.01
(±0.04) (±0.04) (±0.07) (±0.02)
4.68 4.93 4.56 4.27
(±0.30) (±0.28) (±0.50) (±0.63)
1 0 0 1
0.72 / / 0.7
4.26 / / 4.50
0.15 (±0.02) 0.11 (±0.02) 0.12 (±0.03) 0.13 (±0.01)
7.47 7.43 7.90 7.59
(±0.48) (±0.70) (±0.57) (±0.85)
0.1 / / 0.08
7.5 / / 7.89
105
1 2 3 4
12 9 11 15
1.35 1.48 1.47 0.89
(±0.04) (±0.03) (±0.02) (±0.02)
5.42 4.81 4.77 4.76
(±0.38) (±0.48) (±0.46) (±0.41)
0 2 1 0
/ 0.51 (±0.40) 0.67 /
/ 5.27 (±0.22) 4.53 /
0.18 (±0.02) 0.19 (±0.02) 0.18 (±0.03) 0.12 (±0.02)
8.87 9.03 8.65 9.66
(±0.66) (±0.88) (±0.58) (±0.81)
/ 0.15 0.11 /
/ 9.42 (±0.25) 9.12 /
106
1 2 3 4
12 13 13 15
1.23 (±0.04) 0.98 (±0.16) 0.67 (±0.04) 1.01 (±0.03)
5.53 6.03 5.48 5.89
(±0.50) (±0.73) (±0.41) (±0.21)
1 1 1 2
0.88 0.46 0.49 0.24 (±0.45)
5.37 6.12 5.85 6.07 (±0.03)
0.19 (±0.02) 0.17 (±0.02) 0.16 (±0.01) 0.15 (±0.02)
9.98 (±0.61) 10.48 (±1.01) 10.82 (±0.52) 10.53 (±0.68)
0.12 0.1 0.14 0.1 (±0.06)
10.11 10.22 10.48 10.73 (±0.35)
Control
1 2 3 4
11 14 16 14
1.42 1.02 0.99 1.14
0 0 0 0
/ / / /
/ / / /
0.17 (±0.01) 0.15 (±0.01) 0.15 (±0.01) 0.17 (±0.02)
/ / / /
/ / / /
/ / / /
(±0.05) (±0.03) (±0.07) (±0.07)
/ / / /
Average number of bacteria or weight of fetuses or placentas.
Fig. 2. Brucella melitensis infection in pregnant mice. Groups of four pregnant mice were infected with various doses of bacteria intraperitoneally on day 4.5 of gestation. On day 18.5 of gestation, their placentas, spleens, and fetuses were removed and homogenized in saline. The tissue homogenates were plated on TSA plates, and the bacteria recovered from each organ were counted. (A) Spleen vs. placenta (P < 0.05). (B) Living fetus vs. dead fetus.
Z. Wang et al. / The Veterinary Journal 200 (2014) 116–121
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Fig. 3. Brucella melitensis predominantly invades trophoblastic giant cells in the placenta. The placentas not used for bacterial culture were fixed in 10% neutral buffered formalin and routinely processed for histological examination. Specific labeling of B. melitensis in placental sections was performed with anti-Brucella goat serum; replicated bacteria are shown in brown. Normal and infected placentas are shown. (A) 106 CFU-infected placentas; (B) 105 CFU-infected placentas; (C) 104 CFU-infected placentas; (D) 103 CFU-infected placentas; (E) control. Arrows indicate trophoblastic giant cells.
Fig. 4. Brucella melitensis infection in the liver of fetuses. The liver parts not used for bacterial culture were fixed in 10% neutral buffered formalin and routinely processed for histological examination. Specific labeling of B. melitensis in liver sections was performed with anti-Brucella goat serum; replicated bacteria are shown in brown. Control and infected livers are shown. (A) 105 CFU-infected livers; (B) 104 CFU-infected livers; (C) 103 CFU-infected livers; (D) control. Arrows indicate Kupffer cells.
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Fig. 5. Brucella melitensis induces severe placentitis in pregnant mice. (A) In 105 CFU-infected placentas, the cytoplasm of the trophoblastic giant cells was smaller and vacuolated (black arrow), and small foci of coagulative necrosis were observed (red arrow). (B) In 106 CFU-infected placentas, multifocal to coalescing necrosis (arrow) was observed. (C) Normal placenta.
stillborn or autolyzed fetuses were pale, soft, and shrunken, while those supporting living fetuses were dark red, firm, and of expected size, although infected placentas with living fetuses exhibited a yellow rim of material at the periphery. The average weight of the placentas associated with stillborn fetuses was more than 20% lower than that of the placentas supporting living fetuses. In addition to placental damage, splenomegaly, hepatomegaly, and uterine hemorrhage were also evident in the groups infected with doses of 104 CFU and higher. The histological appearance of the placentas from mice infected with 103 or 104 CFU was less severe than that of the placentas from mice infected with 105 or 106 CFU. In the placentas from mice infected with 105 CFU, the cytoplasm of the TGCs was smaller and vacuolated, and small foci of coagulative necrosis within the spongiotrophoblastic zone were observed (Fig. 5A). Following infection with 106 CFU of B. melitensis NI, the recorded lesions were similar to, but more extensive than those observed in mice infected with 105 CFU. The TGCs were heavily infiltrated by neutrophils, and moderate to severe vacuolation and multifocal to coalescing necrosis of the spongiotrophoblastic zone of the placenta was observed (Fig. 5B). Discussion Brucella spp. are facultative intracellular pathogens that cause abortion and infertility in animals. Although mice appear to be resistant to abortion it can be induced at specific time periods, and transmission of virulent B. abortus 544 from the mother to the fetus has been demonstrated in mice (Bosseray and Plommet, 1988). Moreover, previous studies using mouse models have shown that B. abortus specifically replicates in the TGCs (Tobias et al., 1993; Kim et al., 2005) and consequently induces high abortion rates (Kim et al., 2005). However, whether B. melitensis could also infect and induce abortion in pregnant mice, similar to B. abortus, was previously unknown. Therefore, in this study, we investigated the vertical transmission of B. melitensis infection in an ICR mouse model. Oral exposure is usually the natural route of infection for humans and animals. Given the lack of success and technical difficulties associated with the oral route (Pasquali et al., 2003; Paixão et al., 2009), we infected the pregnant mice by IP administration in our studies. This route infects 100% of the mice and induces similar levels of infection for each mouse. In addition, it is technically simpler, allows for large volumes to be used, and is less prone to inoculation errors. Erythritol, a sugar alcohol synthesized in the ungulate placenta, has been shown to stimulate the growth of B. abortus within the placenta of ruminants (Jain et al., 2012; Rodríguez et al., 2012). Although only very low concentrations of erythritol are found in the rodent placenta, the present study indicated that there is preferential growth of B. melitensis in the placentas of mice, as ob-
served for B. abortus. The placenta of each mouse was considered to be an independent unit; however, we observed that all of the placentas in a single mouse were severely infected. Because the placenta itself is an important barrier against infection, once placental infection occurs, the fetus also becomes infected. At necropsy, malformed, autolyzed, and small fetuses were observed in mice infected with doses of 104 CFU or higher. However, no abortions were observed during the experiments, despite the fact that infection with 104 CFU B. abortus has been shown to induce high abortion rates (Kim et al., 2005), indicating that there are differences between the pathogenic characteristics of B. melitensis and B. abortus in mice. Abortion and placental/fetal infection are not always linked. However, in natural hosts, B. abortus infection leads to abortion in cows at late stages of pregnancy due to placental lesions, which are related to bacterial invasion and intracellular replication in trophoblastic cells. Pregnancy leads to a generalized suppression of the adaptive immune system (Weinberg, 1987; Wegmann et al., 1993; Raghupathy, 1997). This immunosuppressed state prevents fetal rejection but has the unfortunate consequence of increasing maternal susceptibility to certain infectious agents (Sano et al., 1986; Krishnan et al., 1996). The uncontrolled growth of Brucella in the placenta may be due to the immune suppression that occurs during pregnancy. Thus, the interactions between abortion and infection with Brucella in animals are extremely complex and still poorly understood. In ruminant brucellosis, erythrophagocytic trophoblasts serve as the initial entry sites for B. abortus into the placenta (Anderson et al., 1986a). In mice, TGCs are phagocytic for a significant portion of gestation and are the most susceptible cells to Brucella infection (Jollie, 1981). In the present study, we observed that the TGCs at the periphery of the mouse placenta served as early sites of bacterial localization and replication, followed by cytoplasmic infection. In infected fetuses, Brucella organisms were also observed in the cytoplasm of Kupffer cells. The degree of colonization and placental damage has been reported to depend on the infectious dose and the type of Brucella strain involved (Bosseray, 1980, 1983). Infection with a 104 CFU or higher dose of B. melitensis caused severe placental damage, characterized by multifocal to coalescing necrosis and heavy infiltration by neutrophils. Experimental infections of B. abortus in pregnant cows also induce necrotizing and suppurative placentitis and the infected fetuses developed fibrinous pleuritis, fibrinous pericarditis and bronchopneumonia (Xavier et al., 2009). Following the necrosis of infected trophoblasts, large numbers of bacteria are released, and the proximity of the fetal capillaries in the ulcerated placenta to the luminal bacteria has been proposed as the source of fetal bacteremia and further placental infection (Anderson et al., 1986a, 1986b). In our study, we did not observed obvious inflammation and typical pathological changes in the fetus of mice.
Z. Wang et al. / The Veterinary Journal 200 (2014) 116–121
Although many vertically transmitted pathogens (bacteria, protozoa and viruses) induce abortion in animals, the mechanisms by which they cause abortion are not well known. In B. abortus, the genes of the dhbCEBA operon have been found to contribute to pathogenicity (Bellaire et al., 2003). In addition, a previous study showed that a transient increase in interferon-c due to Brucella infection contributes to abortion in pregnant mice (Kim et al., 2005), which indicates that complicated immune responses occur during abortion. In the present study, the pregnant mice were observed to develop anti-Brucella antibodies, as did the non-pregnant mice (data not shown). Therefore, it is likely that the particularly high sensitivity of the pregnant animals to brucellosis is not due to a generalized suppression of immunity but instead involves the local suppression of the immune response in the placenta. Conclusions Pregnant ICR mice infected with B. melitensis at a dose of 104 CFU or higher developed multifocal necrotic placentitis. The bacterial load and lesions recorded in the placentas and fetuses increased with the infectious dose, which suggests that this model may be useful for studying the mechanism underlying B. melitensis-induced vertical transmission and for the identification of Brucella abortion-related genes. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgments We thank Professor Ruiping she for her patient guidance on the histological observation, and graduate students Zhaojie Guo and Lingling Chang for their enthusiastic helps in the histological analysis. This work was supported by the National Basic Research Program of China (973 Program; 2010CB530202) and the National Science Foundation of China (Project No. 31372446). References Anderson, T.D., Cheville, N.F., Meador, V.P., 1986a. Pathogenesis of placentitis in the goat inoculated with Brucella abortus. II. Ultrastructural studies. Veterinary Pathology 23, 227–239. Anderson, T.D., Meador, V.P., Cheville, N.F., 1986b. Pathogenesis of placentitis in the goat inoculated with Brucella abortus. I. Gross and histologic lesions. Veterinary Pathology 23, 219–226. Bosseray, N., 1980. Colonization of mouse placentas by Brucella abortus inoculated during pregnancy. British Journal of Experimental Pathology 61, 361–368. Bosseray, N., 1983. Kinetics of placental colonization of mice inoculated intravenously with Brucella abortus at day 15 of pregnancy. British Journal of Experimental Pathology 64, 612–616. Bosseray, N., Plommet, M., 1988. Serum- and cell-mediated immune protection of mouse placenta and fetus against a Brucella abortus challenge: Expression of barrier effect of placenta. Placenta 9, 65–79. Bellaire, B.H., Elzer, P.H., Hagius, S., Walker, J., Baldwin, C.L., Roop, R.M., 2nd, 2003. Genetic organization and iron-responsive regulation of the Brucella abortus 2,3dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild-type virulence in pregnant cattle. Infection and Immunity 71, 1794–1803.
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A pregnant mouse model for the vertical transmission of Brucella melitensis Z. Wang a, S.S. Wang b, G.L. Wang c, T.L. Wu a, Y.L. Lv a, Q.M. Wu a,⇑ a Key Laboratory of Animal Epidemiology and Zoonosis of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Yuanmingyuan Xilu No. 2, Haidian District, Beijing 100193, China b College of Biological Sciences, Anyang Institute of Technology, Anyang 455000, China c College of Veterinary Medicine, Qinghai University, Xining 810000, China
a r t i c l e
i n f o
Article history: Accepted 19 December 2013
Keywords: Brucellosis Bacteriology Histology Pregnant mouse Vertical transmission
a b s t r a c t Abortion is the major clinical sign of brucellosis in animals but little is known about the underlying mechanisms. This study was designed to evaluate a pregnant mouse model for the vertical transmission of Brucella melitensis using four infectious doses: 103 colony-forming units (CFU), 104 CFU, 105 CFU, and 106 CFU. During the experimental period, no instances of abortion were recorded, but stillbirths were observed in the groups infected with doses of 104 CFU and higher. Regardless of whether the fetuses were stillborn or alive, transmission of bacteria to the fetus and bacterial replication in the cytoplasm of placental trophoblast giant cells were detected. A higher degree of bacterial colonization was found in the placenta than in the spleen or fetus. Doses of 105 CFU of B. melitensis or higher produced a severe, necrotizing placentitis similar to the pathological damage observed in ruminants. The data suggest that experimental murine brucellosis resembles ruminant brucellosis and represents a potential model for studying the pathogenic mechanisms of B. melitensis. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
Introduction Brucella spp. are facultative, intracellular, Gram-negative bacteria with a marked affinity for the reproductive tract of pregnant ruminants. It is important to understand the pathogenesis of Brucella infection in pregnant females because of its significant economic impact on ruminant production. Vaginal secretions and aborted fetuses are the most important sources for infection of other animals and also represent an occupational source of infection for humans (Thoen et al., 1993; Neta et al., 2010). During acute brucellosis in pregnant cows, up to 85% of the bacteria are found in cotyledons, placental membranes, and allantoic fluid (Anderson et al., 1986b), resulting in placentitis, fetal death, and abortion (Samartino and Enright, 1993; Tobias et al., 1993). Although the mechanisms underlying placental localization, trophoblast tropism, and abortion associated with ruminant brucellosis are poorly understood, localization of bacteria in chorioallantoic trophoblasts has been shown to be important in the pathogenesis of many reproductive diseases of ruminants. For example, Coxiella burnetii and Chlamydia psittaci have been observed to fill the chorioallantoic trophoblasts of aborted placentas in sheep and goats (Jubb et al., 1985), and Listeria monocytogenes ⇑ Corresponding author. Tel.: +86 10 6273 3901. E-mail address: [email protected] (Q.M. Wu).
initially infects placental villous syncytiotrophoblast (Lecuit et al., 2004). Once Brucella is internalized by the erythrophagocytic trophoblastic epithelial cells from the maternal circulation (Anderson et al., 1986a), the internalized bacteria replicate within the rough endoplasmic reticulum, resulting in secondary infection of adjacent trophoblastic epithelial cells (Anderson et al., 1986a; Meador and Deyoe, 1989). The placenta is known to play an integral role in the pathogenesis of congenital infections and serves as an important barrier against infection. The structure of the bovine placenta is different from that of the murine placenta, but an infectious abortion model in pregnant mice could serve as a useful tool for investigating the mechanisms underlying Brucella pathogenesis. Several studies have addressed the induction of abortion associated with B. abortus infection in pregnant mice (Tobias et al., 1992, 1993; Kim et al., 2005; Watanabe et al., 2008) and have demonstrated transmission of bacteria to the fetus, bacterial replication in the placenta, and high abortion rates (Kim et al., 2005). Although B. abortus is the most important pathogen that induces abortion in pregnant cattle, infection with B. melitensis can also frequently cause placentitis or abortion (Anderson et al., 1986a, 1986b; Meador and Deyoe, 1989; Samadi et al., 2010). In 2007, we isolated a B. melitensis strain from an aborted bovine fetus in the Inner Mongolia region of China, which was designated B. melitensis NI (Liu et al., 2012). Considering the many differences
1090-0233/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2013.12.021
Z. Wang et al. / The Veterinary Journal 200 (2014) 116–121
between B. abortus and B. melitensis, including their biological characteristics, antigenicity, and host selectivity, in addition to the lack of studies addressing the induction of abortion due to B. melitensis infection in pregnant mice, we used in the present study B. melitensis NI to infect pregnant mice. Combined bacteriological and histopathological examinations were performed following the administration of various infective doses to evaluate B. melitensis infection in pregnant ICR mice and to establish an experimental mouse model for the vertical transmission of B. melitensis. Materials and methods Ethics statement All animal research was approved by the Beijing Association for Science and Technology (approval: SYXK, Beijing 2007-0001), and the animal research complied with the Beijing guidelines for laboratory animal welfare and ethics of the Beijing Administration Committee of Laboratory Animals. Bacterial strain and media B. melitensis NI is an epidemic strain that was previously isolated from an aborted bovine fetus from Inner Mongolia in our laboratory (liu et al., 2012). This strain, which is also referred to as the smooth virulent B. melitensis strain biovar 3, induces abortion in pregnant cattle, sheep, and goats. The complete NI genome was sequenced in our laboratory (GenBank accession numbers CP002931 and CP002932). This Brucella strain was routinely grown in tryptic soy broth (TSB) or tryptic soy agar (TSA) at 37 °C. All work with the virulent Brucella strain was performed in biosafety level 3 facilities at China Agricultural University.
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Statistical analysis Student’s t test was applied to analyze the bacteriology data. P < 0.05 was considered significant.
Results B. melitensis NI infection causes stillbirth in pregnant mice at a low infectious dose All 20 mice remained clinically normal for the duration of the experiments based on visual assessment, and no abortion was observed. However, at necropsy, stillbirth, characterized by a lack of heartbeat, or small fetal size and resorbed fetuses, which were malformed or autolyzed, was observed (Fig. 1). The fetuses recorded as resorbed had previously implanted in the uterine wall but were so damaged that they displayed no remaining recognizable fetal structure compared to other fetal units at the same stage of gestation. As shown in Table 1 and Fig. 1, the four mice infected with 106 CFU of B. melitensis NI displayed an average of one or two stillbirths or resorbed fetuses. Following infection with 105 CFU or 104 CFU, 50% of the mice (2/4) exhibited stillbirths. No stillbirths were observed in the group infected with 103 CFU or the control group. However, even in the 106 CFU infection group, one mouse showed a stillbirth rate that was not greater than 30%. These results indicated that B. melitensis NI was pathogenic for pregnant mice, even at a low infectious dose of 104 CFU/mouse.
Mice Female 6- to 10-week-old ICR mice were individually mated with male 6- to 10week-old ICR mice. The day on which a vaginal plug was observed was recorded as day 0.5 of gestation. The normal gestational time for these mice is 19 days. The mice were all purchased from the Weitong Lihua Laboratory Animal Services Centre (Beijing, China), bred in individually ventilated cage rack systems, and subsequently transferred to the biosafety level 3 facilities of China Agricultural University at the beginning of the experiments. All experiments involving animals followed the regulations enacted by the Beijing Administration Office for Laboratory Animals. Experimental infection To study bacterial colonization and the development of placental lesions and abortion, groups of four pregnant mice each were infected intraperitoneally (IP) with 103, 104, 105, or 106 colony-forming units (CFU) of B. melitensis NI in 0.1 mL of phosphate buffered saline (PBS) on day 4.5 of gestation. The exact injection doses expressed in CFU were verified retrospectively via serial dilution, plating, and counting. Four additional mice received 0.1 mL of PBS as a control. On day 18.5 of gestation, all mice were euthanased for bacteriological and histological analyses. Fetal development was evaluated and the fetuses were determined to be alive if there was a heart beat or dead if there was no heart beat (Kim et al., 2005). The minimal infectious dose that caused the proliferation of large numbers of bacteria in placentas and fetuses and induced stillbirth or abortion was determined. Bacteriology The placentas and the corresponding fetuses and spleens of each mouse were removed, weighed, and homogenized in PBS. The tissue homogenates were then serially diluted in PBS, plated on TSA plates, and incubated for 3–5 days at 37 °C under 5% (vol/vol) CO2, and the plates were monitored daily for growth. The bacteria recovered from each organ were counted. B. melitensis colonies were identified based on colony morphology, growth characteristics, and polymerase chain reaction (PCR) analysis using specific primers for B. melitensis as previously described (Huber et al., 2009). Histology The placentas from the mice that were not used for bacterial culture were fixed in 10% neutral buffered formalin and processed for routine histological examination with hematoxylin and eosin (HE) staining. To allow sensitive localization of B. melitensis bacterial colonization in the placental sections, immunohistochemical staining was performed using goat anti-B. melitensis serum as previously reported (Pérez et al., 1998). Non-infected murine placental sections were used as negative controls.
B. melitensis NI predominantly proliferates in the placenta To investigate bacterial multiplication in the infected pregnant mice, we examined the colonization of the B. melitensis NI strain in the spleens, placentas, and corresponding fetuses of the mice. The living or stillborn fetuses and the corresponding placentas of each mouse were analyzed. As shown in Table 1, numerous bacteria were isolated from the placentas of both living and stillborn fetuses in all mice, and the difference between the bacterial counts recorded in the placentas of living and stillborn fetuses was not significant (Table 1, P = 0.5127). In contrast, the bacterial load in the spleen was 2- to 3-log CFU/g lower than that in the placenta of the mice at all tested doses (Fig. 2A, P = 0.004336). Interestingly, both living and dead fetuses were found to be infected with B. melitensis NI, and the bacterial load was not significantly different between the living and dead fetuses (Fig. 2B, P = 0.8273). However, the number of bacteria recovered from the placentas, spleens, and fetuses in each group was positively correlated with the infectious dose (Fig. 2A and B). Immunostaining of bacterially infected placental specimens from each infection group revealed that B. melitensis was present in numerous trophoblast giant cells (TGCs), especially in the placentas of mice infected with 105 or 106 CFU, and the cytoplasm of the bacterially infected trophoblasts contained large numbers of B. melitensis surrounding cell nuclei. Furthermore, we observed intact TGCs exhibiting brown staining within the cytoplasm, indicating the presence of intracellular Brucella (Fig. 3). Although bacteria were also detected in the cytoplasm of fetal Kupffer cells (Fig. 4), the positive staining signal was much weaker than that observed in the placenta. Regardless of the location, the positive staining signal for Brucella was dose dependent. B. melitensis NI induces severe placentitis in pregnant mice Among the Brucella-infected mice, gross placental lesions were observed in mice receiving doses of 104 CFU or higher. The placentas of these mice varied in size and color. Those associated with
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Fig. 1. Brucella melitensis NI infection caused fetal resorption and stillbirth in pregnant mice. Resorbed fetuses were characterized as malformed (A) or autolyzed (B); stillbirths were characterized by retarded growth (C), the lack of a heart beat, or small fetus size (D). Normal fetus (E).
Table 1 Bacteriological results for Brucella melitensis NI-infected pregnant mice. Dose/ mouse
Mouse Number
Fetus
Number
a
Placenta
Living Weight (g)a
Dead
Living
Bacterial number (log)a
Number
Weight (g)a
Bacterial number (log)a
Weight (g)a
Dead
Bacterial number (log)a
Weight (g)a
Bacterial number (log)a
103
1 2 3 4
12 14 17 16
1.16 1.04 0.91 0.92
(±0.04) (±0.05) (±0.04) (±0.03)
2.47 (±0.28) 2.91 (±0.22) 2.89 (±0.10) 2.77(±0.37)
0 0 0 0
/ / / /
/ / / /
0.14 (±0.01) 0.13 (±0.02) 0.14 (±0.01) 0.11 (±0.02)
5.63 5.82 6.04 6.49
(±0.56) (±0.74) (±0.65) (±0.74)
/ / / /
/ / / /
104
1 2 3 4
15 14 13 13
0.92 0.97 0.98 1.01
(±0.04) (±0.04) (±0.07) (±0.02)
4.68 4.93 4.56 4.27
(±0.30) (±0.28) (±0.50) (±0.63)
1 0 0 1
0.72 / / 0.7
4.26 / / 4.50
0.15 (±0.02) 0.11 (±0.02) 0.12 (±0.03) 0.13 (±0.01)
7.47 7.43 7.90 7.59
(±0.48) (±0.70) (±0.57) (±0.85)
0.1 / / 0.08
7.5 / / 7.89
105
1 2 3 4
12 9 11 15
1.35 1.48 1.47 0.89
(±0.04) (±0.03) (±0.02) (±0.02)
5.42 4.81 4.77 4.76
(±0.38) (±0.48) (±0.46) (±0.41)
0 2 1 0
/ 0.51 (±0.40) 0.67 /
/ 5.27 (±0.22) 4.53 /
0.18 (±0.02) 0.19 (±0.02) 0.18 (±0.03) 0.12 (±0.02)
8.87 9.03 8.65 9.66
(±0.66) (±0.88) (±0.58) (±0.81)
/ 0.15 0.11 /
/ 9.42 (±0.25) 9.12 /
106
1 2 3 4
12 13 13 15
1.23 (±0.04) 0.98 (±0.16) 0.67 (±0.04) 1.01 (±0.03)
5.53 6.03 5.48 5.89
(±0.50) (±0.73) (±0.41) (±0.21)
1 1 1 2
0.88 0.46 0.49 0.24 (±0.45)
5.37 6.12 5.85 6.07 (±0.03)
0.19 (±0.02) 0.17 (±0.02) 0.16 (±0.01) 0.15 (±0.02)
9.98 (±0.61) 10.48 (±1.01) 10.82 (±0.52) 10.53 (±0.68)
0.12 0.1 0.14 0.1 (±0.06)
10.11 10.22 10.48 10.73 (±0.35)
Control
1 2 3 4
11 14 16 14
1.42 1.02 0.99 1.14
0 0 0 0
/ / / /
/ / / /
0.17 (±0.01) 0.15 (±0.01) 0.15 (±0.01) 0.17 (±0.02)
/ / / /
/ / / /
/ / / /
(±0.05) (±0.03) (±0.07) (±0.07)
/ / / /
Average number of bacteria or weight of fetuses or placentas.
Fig. 2. Brucella melitensis infection in pregnant mice. Groups of four pregnant mice were infected with various doses of bacteria intraperitoneally on day 4.5 of gestation. On day 18.5 of gestation, their placentas, spleens, and fetuses were removed and homogenized in saline. The tissue homogenates were plated on TSA plates, and the bacteria recovered from each organ were counted. (A) Spleen vs. placenta (P < 0.05). (B) Living fetus vs. dead fetus.
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Fig. 3. Brucella melitensis predominantly invades trophoblastic giant cells in the placenta. The placentas not used for bacterial culture were fixed in 10% neutral buffered formalin and routinely processed for histological examination. Specific labeling of B. melitensis in placental sections was performed with anti-Brucella goat serum; replicated bacteria are shown in brown. Normal and infected placentas are shown. (A) 106 CFU-infected placentas; (B) 105 CFU-infected placentas; (C) 104 CFU-infected placentas; (D) 103 CFU-infected placentas; (E) control. Arrows indicate trophoblastic giant cells.
Fig. 4. Brucella melitensis infection in the liver of fetuses. The liver parts not used for bacterial culture were fixed in 10% neutral buffered formalin and routinely processed for histological examination. Specific labeling of B. melitensis in liver sections was performed with anti-Brucella goat serum; replicated bacteria are shown in brown. Control and infected livers are shown. (A) 105 CFU-infected livers; (B) 104 CFU-infected livers; (C) 103 CFU-infected livers; (D) control. Arrows indicate Kupffer cells.
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Fig. 5. Brucella melitensis induces severe placentitis in pregnant mice. (A) In 105 CFU-infected placentas, the cytoplasm of the trophoblastic giant cells was smaller and vacuolated (black arrow), and small foci of coagulative necrosis were observed (red arrow). (B) In 106 CFU-infected placentas, multifocal to coalescing necrosis (arrow) was observed. (C) Normal placenta.
stillborn or autolyzed fetuses were pale, soft, and shrunken, while those supporting living fetuses were dark red, firm, and of expected size, although infected placentas with living fetuses exhibited a yellow rim of material at the periphery. The average weight of the placentas associated with stillborn fetuses was more than 20% lower than that of the placentas supporting living fetuses. In addition to placental damage, splenomegaly, hepatomegaly, and uterine hemorrhage were also evident in the groups infected with doses of 104 CFU and higher. The histological appearance of the placentas from mice infected with 103 or 104 CFU was less severe than that of the placentas from mice infected with 105 or 106 CFU. In the placentas from mice infected with 105 CFU, the cytoplasm of the TGCs was smaller and vacuolated, and small foci of coagulative necrosis within the spongiotrophoblastic zone were observed (Fig. 5A). Following infection with 106 CFU of B. melitensis NI, the recorded lesions were similar to, but more extensive than those observed in mice infected with 105 CFU. The TGCs were heavily infiltrated by neutrophils, and moderate to severe vacuolation and multifocal to coalescing necrosis of the spongiotrophoblastic zone of the placenta was observed (Fig. 5B). Discussion Brucella spp. are facultative intracellular pathogens that cause abortion and infertility in animals. Although mice appear to be resistant to abortion it can be induced at specific time periods, and transmission of virulent B. abortus 544 from the mother to the fetus has been demonstrated in mice (Bosseray and Plommet, 1988). Moreover, previous studies using mouse models have shown that B. abortus specifically replicates in the TGCs (Tobias et al., 1993; Kim et al., 2005) and consequently induces high abortion rates (Kim et al., 2005). However, whether B. melitensis could also infect and induce abortion in pregnant mice, similar to B. abortus, was previously unknown. Therefore, in this study, we investigated the vertical transmission of B. melitensis infection in an ICR mouse model. Oral exposure is usually the natural route of infection for humans and animals. Given the lack of success and technical difficulties associated with the oral route (Pasquali et al., 2003; Paixão et al., 2009), we infected the pregnant mice by IP administration in our studies. This route infects 100% of the mice and induces similar levels of infection for each mouse. In addition, it is technically simpler, allows for large volumes to be used, and is less prone to inoculation errors. Erythritol, a sugar alcohol synthesized in the ungulate placenta, has been shown to stimulate the growth of B. abortus within the placenta of ruminants (Jain et al., 2012; Rodríguez et al., 2012). Although only very low concentrations of erythritol are found in the rodent placenta, the present study indicated that there is preferential growth of B. melitensis in the placentas of mice, as ob-
served for B. abortus. The placenta of each mouse was considered to be an independent unit; however, we observed that all of the placentas in a single mouse were severely infected. Because the placenta itself is an important barrier against infection, once placental infection occurs, the fetus also becomes infected. At necropsy, malformed, autolyzed, and small fetuses were observed in mice infected with doses of 104 CFU or higher. However, no abortions were observed during the experiments, despite the fact that infection with 104 CFU B. abortus has been shown to induce high abortion rates (Kim et al., 2005), indicating that there are differences between the pathogenic characteristics of B. melitensis and B. abortus in mice. Abortion and placental/fetal infection are not always linked. However, in natural hosts, B. abortus infection leads to abortion in cows at late stages of pregnancy due to placental lesions, which are related to bacterial invasion and intracellular replication in trophoblastic cells. Pregnancy leads to a generalized suppression of the adaptive immune system (Weinberg, 1987; Wegmann et al., 1993; Raghupathy, 1997). This immunosuppressed state prevents fetal rejection but has the unfortunate consequence of increasing maternal susceptibility to certain infectious agents (Sano et al., 1986; Krishnan et al., 1996). The uncontrolled growth of Brucella in the placenta may be due to the immune suppression that occurs during pregnancy. Thus, the interactions between abortion and infection with Brucella in animals are extremely complex and still poorly understood. In ruminant brucellosis, erythrophagocytic trophoblasts serve as the initial entry sites for B. abortus into the placenta (Anderson et al., 1986a). In mice, TGCs are phagocytic for a significant portion of gestation and are the most susceptible cells to Brucella infection (Jollie, 1981). In the present study, we observed that the TGCs at the periphery of the mouse placenta served as early sites of bacterial localization and replication, followed by cytoplasmic infection. In infected fetuses, Brucella organisms were also observed in the cytoplasm of Kupffer cells. The degree of colonization and placental damage has been reported to depend on the infectious dose and the type of Brucella strain involved (Bosseray, 1980, 1983). Infection with a 104 CFU or higher dose of B. melitensis caused severe placental damage, characterized by multifocal to coalescing necrosis and heavy infiltration by neutrophils. Experimental infections of B. abortus in pregnant cows also induce necrotizing and suppurative placentitis and the infected fetuses developed fibrinous pleuritis, fibrinous pericarditis and bronchopneumonia (Xavier et al., 2009). Following the necrosis of infected trophoblasts, large numbers of bacteria are released, and the proximity of the fetal capillaries in the ulcerated placenta to the luminal bacteria has been proposed as the source of fetal bacteremia and further placental infection (Anderson et al., 1986a, 1986b). In our study, we did not observed obvious inflammation and typical pathological changes in the fetus of mice.
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Although many vertically transmitted pathogens (bacteria, protozoa and viruses) induce abortion in animals, the mechanisms by which they cause abortion are not well known. In B. abortus, the genes of the dhbCEBA operon have been found to contribute to pathogenicity (Bellaire et al., 2003). In addition, a previous study showed that a transient increase in interferon-c due to Brucella infection contributes to abortion in pregnant mice (Kim et al., 2005), which indicates that complicated immune responses occur during abortion. In the present study, the pregnant mice were observed to develop anti-Brucella antibodies, as did the non-pregnant mice (data not shown). Therefore, it is likely that the particularly high sensitivity of the pregnant animals to brucellosis is not due to a generalized suppression of immunity but instead involves the local suppression of the immune response in the placenta. Conclusions Pregnant ICR mice infected with B. melitensis at a dose of 104 CFU or higher developed multifocal necrotic placentitis. The bacterial load and lesions recorded in the placentas and fetuses increased with the infectious dose, which suggests that this model may be useful for studying the mechanism underlying B. melitensis-induced vertical transmission and for the identification of Brucella abortion-related genes. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgments We thank Professor Ruiping she for her patient guidance on the histological observation, and graduate students Zhaojie Guo and Lingling Chang for their enthusiastic helps in the histological analysis. This work was supported by the National Basic Research Program of China (973 Program; 2010CB530202) and the National Science Foundation of China (Project No. 31372446). References Anderson, T.D., Cheville, N.F., Meador, V.P., 1986a. Pathogenesis of placentitis in the goat inoculated with Brucella abortus. II. Ultrastructural studies. Veterinary Pathology 23, 227–239. Anderson, T.D., Meador, V.P., Cheville, N.F., 1986b. Pathogenesis of placentitis in the goat inoculated with Brucella abortus. I. Gross and histologic lesions. Veterinary Pathology 23, 219–226. Bosseray, N., 1980. Colonization of mouse placentas by Brucella abortus inoculated during pregnancy. British Journal of Experimental Pathology 61, 361–368. Bosseray, N., 1983. Kinetics of placental colonization of mice inoculated intravenously with Brucella abortus at day 15 of pregnancy. British Journal of Experimental Pathology 64, 612–616. Bosseray, N., Plommet, M., 1988. Serum- and cell-mediated immune protection of mouse placenta and fetus against a Brucella abortus challenge: Expression of barrier effect of placenta. Placenta 9, 65–79. Bellaire, B.H., Elzer, P.H., Hagius, S., Walker, J., Baldwin, C.L., Roop, R.M., 2nd, 2003. Genetic organization and iron-responsive regulation of the Brucella abortus 2,3dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild-type virulence in pregnant cattle. Infection and Immunity 71, 1794–1803.
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