**3. Preclinical models of Brucellar spondylodiscitis**

Mice, rats, rabbits, and sheep represent the available candidates to induce Brucellosis [23–26]. However, the preclinical model of Brucellar spondylodiscitis with a possible high translational efficiency has only been established in rabbits so far [14].

#### **3.1 Mice**

Mice are the most extensively used to investigate chronic infections of *Brucella* [15]. Enright and colleagues constructed the Brucellosis model with the mouse by intravenous injection of 5 × 104 colony forming units (CFU) of either *Brucella* attenuated strain 19 or *Brucella abortus* strain 2308 to confirm *Brucella abortus* produces a chronic granulomatous response in mice, and extend earlier studies in demonstrating that prominent acute and subacute inflammatory responses also occur [27]. Steven *et al.* made the mouse model with the method of intraperitoneal injections of 105 CFU of *Brucella* strain 2308 or 107 CFU of either strain 19 or RB51 [28]. Tobias' Brucellosis mouse model was built by intraperitoneally infected with 106 CFU of *Brucella abortus* strain 2308 [29]. The spleen is the most heavily colonized organ with voluminous histiocytic infiltrates and multifocal microgranulomas [27–29]. Besides the spleen, the liver is also a common site for colonization and replication of *Brucella* in the mice [27, 30, 31]. Mice infected with virulent strains of *Brucella* have mild to moderate hepatitis characterized by neutrophilic infiltrate at early stages of the infection, followed by histiocytic infiltrate with epithelioid cells and microgranulomas at its chronic stages [27, 29]. It is noteworthy that *Brucella* infection in mice results in lesions that mimic those described in chronic infections

in humans, and patients with chronic Brucellosis may develop splenomegaly and hepatomegaly [32]. In addition, multifocal granulomas with epithelioid macrophages are also detected in the liver or spleen parenchyma of patients who were infected with *Brucella* [33, 34]. Chronic infection of the *Brucella* in humans may also yield osteoarticular alterations, including osteoarthritis and spondylodiscitis [35]. Rajashekara *et al.* reported that mice may develop bacterial colonization in periarticular tissues during the chronic stages of *Brucella melitensis* infection [36]. In mice surviving over 45 days after intraperitoneal infection of *Brucella melitensis*, the bioluminescent *Brucella melitensis* were detected in the vertebral joints of their tails, suggesting that the mice might be a useful model for the study of human osteoarticular diseases, including the Brucellar spondylodiscitis [16]. Comparisons of disease manifestations (e.g. timing of the disease onset, structural alterations of the spines, and definite localization of the bacterial foci) between animal and human Brucellar spondylodiscitis would be beneficial for evaluating its potential translational values and further recognizing specific histological, radiographic, and clinical signs, allowing for an early detection and intervention of human Brucellar spondylodiscitis.

The mouse model has been utilized for the evaluation of the efficiency of different pharmacotherapies for human Brucellosis [37–39]. Several lines of evidence suggest that mice treated with ciprofloxacin, by subcutaneous (40 mg/kg), digestive (200 mg/kg), or intraperitoneal (20 mg/kg) route, are not able to control the infection of *Brucella* melitensis. In contrast, mice treated with doxycycline (40 mg/kg) at 24 hours after infection efficiently clear the infection [38, 39]. Shasha *et al.* showed that mice administered intraperitoneally with doxycycline (40 mg/kg/day) or rifampin (25 mg/kg/day) had high levels of antibiotics in the blood following 1-hour postinjection (doxycycline: 5.4 μg/ml and rifampin: 18 μg/ml, respectively) and were able to clear the infection. The treatment regimen of the usage of doxycycline and rifampin is consistent with the therapeutic protocols of human Brucellosis.

#### **3.2 Rats**

The rats, more resistant to *Brucella* infection than the mice [15], can develop the persistent bacteremia, and do not have a spontaneous cure over the 1-month infection [23]. Therefore, rats can serve as a model candidate to evaluate the increased susceptibility to *Brucella* infection, which mimics the chronic symptoms in patients. Yumuk *et al*. analyzed the rat model by intraperitoneal injection of 2 × 105 to 4 × 105 CFU of *Brucella melitensis* strain 16 M [23]. Moreover, the rat model has also been used to analyze the efficacy of various antibiotics to treat Brucellosis [40–42] and to evaluate the pathological properties and clinical characters of *Brucella* infection during the pregnancy [43]. Also, the similar morphology of the spine at the axial plane between rats and humans supports their application as an applicable candidate for the spine research [19, 44]. Since patients with Brucellar spondylodiscitis are commonly detected at the late phase of the disease, rats, as the potential model of chronic Brucellar spondylodiscitis, may be beneficial to assist to exam specific remedies against those chronic manifestations within a clinically relevant setting.

The usefulness of pharmacotherapy has been investigated in the treatment for Brucellosis in the rat model. Geyik *et al*. reported that rats were administered orally with rifampicin (50 mg/kg/day) and doxycycline (40 mg/kg/day), or spiramycin (50 mg/kg/day), or a combination of spiramycin and rifampicin at the same dose for 21 days [40]. All the rats were cured with the treatment results that the effectivities of spiramycin and rifampicin plus spiramycin were similar to rifampicin plus

*Preclinical Models of Brucellar Spondylodiscitis DOI: http://dx.doi.org/10.5772/intechopen.98754*

doxycycline. This result is helpful for the effective alternative in the treatment of human Brucellosis. Furthermore, Sezak and colleagues proposed that moxifloxacin might be an alternative choice in the treatment of Brucellosis [42]. Doxycycline (10 mg/kg/day) plus rifampicin (6 mg/kg/day) were administered intragastrically in Yumuk's study [41]. In the experiment group, all the rats received a liquid diet containing ethanol. The cure rate was 64.71% in ethanol-fed and 100% in ethanol free group. The results suggest that ethanol ingestion diminishes the efficacy of doxycycline plus rifampicin combination therapy of rat Brucellosis model, which holds implications for the treatment programme for human Brucellosis.

## **3.3 Rabbits**

Compared with other animals, rabbits are medium-sized animals which frequently used in spine research with various advantages [45]. The rabbit spine maintains considerable morphological and structural similarities to the human spine, and its body size allows for an adequate exposure during the surgical interventions [46, 47]. Furthermore, rabbits yield higher possibilities than rodents to successfully translate preclinical discoveries into humans [48, 49]. Similarly, compared with larger animals, radiographic analyses are largely convenient in rabbits particularly for *in vivo* explorations [50].

Age is a critical issue to be considered when establishing a rabbit model of local restricted Brucellar spondylodiscitis. The significant variance of the innate immune response between young and adult rabbits against infections of foreign microbes should be recognized [14]. Virus related studies highlighted the significantly superior innate immune system in young rabbits (<4 weeks) over adult rabbits, which contributed to their distinct susceptibility to virus infections [51–53]. These data may partially explain the fact that adult rabbits are only partially susceptible to *Brucella* infection [15], and about 20% of infected animals developed a very short and sporadic bacteremia [25]. In contrast, our previous experiment within young rabbits showed that 83.4% (10 out of 12) rabbits were successfully infected by the intraosseous injection of 3 × 107 CFU of *Brucella melitensis* strain M5–90 [14]. Moreover, to mimic a local restricted inflammation without systemic dissemination of the microbes, studies via a local injection intervention with a reduced dose within young rabbits can avoid the unanticipated animal death due to the local or systematic administration with a relatively larger dosage within adult rabbits. Therefore, even with a relatively smaller size of the spine than adult rabbits, young rabbits may be more suitable to establish such a local restricted model of Brucellar spondylodiscitis.

Of note, despite the animal model for Brucellar spondylodiscitis has been established in rabbits, no studies about its treatments are available for the rabbit Brucellosis.

#### **3.4 Sheep**

Although *Brucella* ovis is one of the few classical *Brucella* species that do not have zoonotic potential, this organism is considered a major cause of reproductive failure in sheep [54]. The attenuated vaccine strain *Brucella melitensis* Rev.1, against *Brucella* infection of sheep and goats, are still the most efficient ones available among living vaccines [55]. When the bacteria are administered by the classic subcutaneous method (109 – 2 × 109 CFU) in the sheep, a long-lasting serologic response is subsequently yielded. Primary manifestations of *Brucella* ovis infection in sheep are lesions of the epididymis and testis in males (e.g., epididymitis and orchitis), placentitis and abortion in ewes, and occasionally perinatal death

in lambs [56], as well as arthritis [57]. The sheep spine is relatively larger than humans, particularly in vertebral body width, which would be advantageous for the easier surgical operation [22]. However, sheep are expensive to house and the operation requires special settings, which also hinder the widely accessibility of this model [58].

Oxytetracycline combined with streptomycin were evaluated for eliminating *Brucella melitensis* from naturally infected sheep. The following treatment regiments were equally effective in eliminating *Brucella* in the sheep: oxytetracycline (20 mg/kg/day) intravenously daily for 6 weeks combined with streptomycin (20 mg/kg/day) intramuscularly for 3 weeks; long-acting oxytetracycline 20 mg/kg intramuscularly every 3 days for 6 weeks plus streptomycin 20 mg/kg intramuscularly every 3 days for 3 weeks; long-acting oxytetracycline mg/kg intramuscularly every 3 days for 6 weeks combined with streptomycin 20 mg/kg intramuscularly every 3 days for 3 weeks [59]. The data indicated the most effective and practical regimen for eliminating *Brucella* in the sheep.
