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Investigation of the epidemiology, pathogenicity and immunogenicity of Bordetella bronchiseptica isolated from cats and dogs in China from 2021 to 2023

Animal Diseases volume 4, Article number: 16 (2024) Cite this article

Abstract

Bordetella bronchiseptica (Bb) is recognized as a leading cause of respiratory diseases in dogs and cats. However, epidemiological data on Bb in dogs and cats in China are still limited, and there is no commercially available vaccine. Live vaccines containing Bb that are widely used abroad are generally effective but can establish latency and potentially reactivate to cause illness in some immunodeficient vaccinated recipients, raising safety concerns. In this study, 34 canine-derived and two feline-derived Bb strains were isolated from 1809 canine and 113 feline nasopharyngeal swab samples collected from eight provinces in China from 2021 to 2023. The PCR results showed that the percentage of positive Bb was 22.94% (441/1922), and more than 90% of the Bb isolates had four virulence factor-encoding genes (VFGs), namely, fhaB, prn, betA and dnt. All the isolated strains displayed a multidrug-resistant phenotype. The virulence of 10 Bb strains isolated from dogs with respiratory symptoms was tested in mice, and we found that eight isolates were highly virulent. Furthermore, the eight Bb isolates with high virulence were inactivated and intramuscularly injected into mice, and three Bb strains (WH1218, WH1203 and WH1224) with the best protective efficacy were selected. Dogs immunized with these three strains exhibited strong protection against challenge with the Bb field strain WH1218. Ultimately, the WH1218 strain with the greatest protection in dogs was selected as the vaccine candidate. Dogs and cats that received a vaccine containing 109 CFU of the inactivated WH1218 strain showed complete protection against challenge with the Bb field strain WH1218. This study revealed that Bb is an important pathogen that causes respiratory diseases in domestic dogs and cats in China, and all the isolates exhibited multidrug resistance. The present work contributes to the current understanding of the prevalence, antimicrobial resistance, and virulence genes of Bb in domestic dogs and cats. Additionally, our results suggest that the WH1218 strain is a promising candidate safe and efficacious inactivated Bb vaccine.

Introduction

Canine infectious respiratory disease (CIRDC) is a worldwide epidemic syndrome involving multiple viral and bacterial pathogens (Day et al. 2020; Fastrès et al. 2020; Maboni et al. 2019; Matsuu et al. 2020). Bordetella bronchiseptica (Bb) is one of the major pathogenic bacteria for CIRDC and can also act as a single pathogen (Bemis et al. 1977a; Chambers et al. 2019; Maboni et al. 2019). Bb is a gram-negative bacillus that colonizes the respiratory tract of mammals, and it is susceptible to nearly all warm-blooded animals (Goodnow 1980; Woolfrey and Moody 1991). This pathogen is the causative agent of kennel cough in dogs and suppurative bronchopneumonia in cats (Speakman et al. 1999). Furthermore, previous studies have demonstrated that Bb can infect humans (Goodnow 1980; Woolfrey and Moody 1991), especially immunocompromised patients who are at increased risk of infection (Agarwal et al. 2022; Gupta et al. 2019; Sameed et al. 2019; Tatem et al. 2023). Moreover, coinfections of SARS-CoV-2 and Bb have been reported (Faqihi et al. 2020; Mulkoju et al. 2022; Nagarakanti and Bishburg 2021; Papantoniou et al. 2021; Pierce et al. 2022). Like other members of the Bordetella genus, Bb can produce several important virulence factors, including filamentous hemagglutinin, protein toxin, adenylyl cyclase toxin, skin necrosis toxin, and a type III secretion system and effector proteins involved in its pathogenesis (Kamanova 2020; Linz et al. 2016), and it has been reported that Bordetella utilizes a type 3 secretion system to manipulate VIP/VPAC2 signaling to promote colonization and persistence of three classical Bordetella species in the lower respiratory tract (First et al. 2023). Porter et al. showed that this pathogen can grow in lake water, which suggests that Bb may be a free-living pathogen (Porter et al. 1991; Porter and Wardlaw 1993). It is a great challenge to determine the exact infection rate of Bb; it usually takes more than three months to completely eradicate the pathogen after infection (Bemis et al. 1977b), and many infected animals remain asymptomatic (Pecora 1976). Aerosol and direct contact facilitate the transmission of Bb, and a previous study reported a transmission rate of 100% by these two means from experimentally infected dogs to fully susceptible dogs within only 1–4 d (McCandlish et al. 1978b; Thompson et al. 1976).

Currently, there is no commercial vaccine against Bb in dogs and cats in China. Vaccine strains have been reported to survive in dogs for at least two weeks after immunization with live attenuated vaccines widely used abroad (Shade and Goodnow 1979). In addition, the possibility of virulence reactivation (from avirulent to virulent) appears to be low, but there is still potential for compromised hosts to be clinically significantly infected (Ostle 1989). Furthermore, vaccine-derived Bb has been reported to be transmissible from dogs to immunocompromised patients (Doo et al. 2017; Kraai et al. 2023; Yacoub et al. 2014). In veterinary clinics across the U.S., the median canine Bb vaccination rate is as high as 68.7%; in cats, it ranges from 1–100% (Malter et al. 2022). Therefore, it is necessary to develop a vaccine with good safety and protective efficacy against the prevalent strain of Bb in China. Inactivated vaccines have long been considered safe and have exhibited some effectiveness (Abdoli et al. 2022; Jin et al. 2022; Marouf et al. 2022). The group at the University of Glasgow performed a series of studies in the mid-to-late 1970s and provided proof of the principle that parenteral vaccines could stimulate Bb disease-sparing responses (McCandlish et al. 1978a, b). In this study, canine and feline nasopharyngeal swab samples were collected to isolate Bb strains, and the drug susceptibility and virulence factor-encoding genes (VFGs) of the Bb isolates were evaluated. The virulence of strains isolated from samples with respiratory symptoms was evaluated in mice. Next, the protective efficacy of virulent isolates was compared in mice and dogs, and WH1218, which has the best protection in dogs, was selected. Dogs and cats receiving a vaccine containing 109 CFU of the inactivated WH1218 strain showed complete protection. This study indicated that Bb is prevalent in dogs and cats in parts of China and that the WH1218 strain is a promising candidate as a safe and efficacious inactivated Bb vaccine.

Results

Epidemiological investigation of Bb in China from 2021 to 2023

Across different provinces in China, the positivity rates varied between 15.47% and 36.23%, the isolation rates ranged between 0% and 4.48% (Fig. 1A, B), and the overall separation rate was 1.87% (36/1922). Thirty-four strains were isolated from 1809 canine nasopharyngeal swabs, and two strains were isolated from 113 feline nasopharyngeal swabs. The positive rate and isolation rate of dog samples were 23.88% (432/1809) and 1.88% (34/1809), respectively, both of which were greater than those of cat samples (7.96%, 9/113 and 1.77%, 2/113). Furthermore, the percentage of samples isolated from dogs with respiratory symptoms was 6.54% (10/153), which was greater than that from dogs without respiratory symptoms (1.45%, 24/1656) (Fig. 1C). The PCR products of the Bb-positive colonies had a specific band at 237 bp on an agarose gel and showed grayish-white, smooth, raised colonies after 28-44 h of incubation on a TSA plate (Fig. 1D). Biochemical experiments revealed that the isolated Bb strains were unable to ferment various sugars, such as fructose, glucose, mannitol, maltose, sorbose and lactose. The methyl red (MR), Voges-Proskauer (VP), and indole reactions were all negative, while the oxidase, urease and hydrogen peroxide enzyme reactions were positive (data not shown).

Fig. 1
figure 1

Isolation of Bb (B. bronchiseptica) from nasopharyngeal swab samples collected from domestic dogs and cats in various regions of China from 2021 to 2023. A The geographic locations of sample collection are represented on the map, with numbers indicating the ratio of Bb-positive samples to the total number of samples collected from each province. B The percentage of Bb-positive nasopharyngeal swabs collected from domestic dogs and cats in select regions of China during the period from 2021 to 2023. C Positivity rate and isolation rate of Bb from nasopharyngeal swab samples collected from dogs and cats. D PCR results of isolates and characterization of colonies on TSA plates

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Drug resistance analysis of Bb isolates

The antimicrobial susceptibility evaluation results revealed that 100% of the isolated strains (n = 36) were sensitive to amikacin, gentamicin, kanamycin, doxycycline, florfenicol, enrofloxacin, ciprofloxacin, chloromycetin, and polymyxin B. Over 80% of the isolated strains were sensitive to tetracycline (34 strains, 99.45%) and norfloxacin (32 strains, 88.98%). Approximately 55.56% (n = 20) of the strains were sensitive to streptomycin, 41.67% (n = 15) to ceftriaxone, and fewer proportions of the strains were sensitive to rifampicin (27.78%, n = 10), cefotaxime (13.89%, n = 5), selectrin (13.89%, n = 5), and ampicillin (8.33%, n = 3) (Fig. 2A, Table 1). All the strains (n = 36) were resistant to fosfomycin. In addition, 100% of the isolated strains (n = 36) were resistant to at least three antibiotics, 72.22% (n = 26) to at least four, 50% (n = 18) to at least five, and 22.22% (n = 8) to at least six (Fig. 2B). All the feline-derived strains were resistant to the three drugs (Fig. 2C).

Fig. 2
figure 2

Drug resistance phenotypes of 36 Bb isolates from dogs and cats in parts of China. A Percentages of susceptible, intermediate and resistant isolates to the 18 evaluated antibiotics. B Distribution of multidrug resistance. Ten Bb strains (eight dog-derived and two cat-derived Bb strains) exhibited resistance to three antibiotics, and 26 Bb strains displayed resistance to more than three antibiotics. C Number of isolates with multidrug resistance. The X-axis shows the number of Bb strains, and the Y-axis shows multidrug resistance. AMK, amikacin; GEN, gentamicin; KAN, kanamycin; CRO, ceftriaxone; CTX, cefotaxime; DOX, doxycycline; SM, streptomycin; SXT, Selectrin; ENR, enrofloxacin; CIP, ciprofloxacin; CHL, chloramphenicol; FLO, florfenicol; RIF, amoxicillin; AMP, ampicillin; NOR, norfloxacin; FOS, fosfomycin; TET, tetracycline; PMB, polymyxin B

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Table 1 Drug resistance phenotypes of 36 Bb isolates from dogs and cats in parts of China
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The antibiotics tested in this study can be categorized into nine groups: aminoglycosides (AMK, GEN, KAN and SM), broad-spectrum cephalosporins (CRO and CTX), fluoroquinolones (CIP, ENR, NOR and RIF), phenols (CHL and FLO), penicillins (AMP), tetracyclines (TET and DOX), polymyxins (PMB), sulfonamide antibiotics (SXT) and polyphosphates (FOS). All Bordetella bronchiseptica isolates in this study (100%, n = 36) were resistant to at least three classes of antibiotics. These 36 Bb isolates could be defined as multidrug-resistant (MDR) strains according to international expert recommendations for provisional standard definitions of acquired resistance (Magiorakos et al. 2012). Among these multidrug-resistant strains, 33.33% (n = 12), 22.22% (n = 8), 27.78% (n = 10), and 16.67% (n = 6) of the isolates were resistant to class III, IV, V and VI drugs, respectively.

Analysis of the virulence factor-encoding genes of Bb isolates

The positivity rates of the fhaB, prn, cyaA, dnt and betA genes were 100% (n = 36), 100% (n = 36), 22.22% (n = 8), 91.67% (n = 33), and 100% (n = 36), respectively (Fig. 3A, B). Furthermore, at least three out of five types of VFGs in all 36 strains were detected as positive. A total of 19.44% of the isolated strains (n = 7) were found to simultaneously contain fhaB, prn, cyaA, dnt and betA (Fig. 3C). The remaining strains contained the following 3 VFG combinations: “fhaB + prn + dnt + betA” (72.22%, n = 26), “fhaB + prn + cyaA + betA” (2.78%, n = 1), and “fhaB + prn + betA” (5.56%, n = 2) (Fig. 3C).

Fig. 3
figure 3

PCR detection of virulence factor-encoding genes (VFGs) from Bb isolates. A Agarose gel analysis of the betA (band 2, 474 bp), fhaB (band 3, 475 bp), cyaA (band 1, 377 bp), dnt (band 4, 491 bp), and prn (band 5, 555 bp) PCR products. B Positive rate of 5 VFGs. C Number of strains containing different VFGs. X-axis: Number of Bb strains; Y-axis: Different VFGs

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Pathogenicity evaluation of Bb isolates in mice

Ten strains isolated from dogs with respiratory symptoms were injected intraperitoneally at 107 CFU/mouse, and the results showed that eight strains were pathogenic, while two strains were not. The fatality rate of strains WH1218 and WH1201 was 100% (Table 2), and the fatality rate of WH1218 was greater than that of WH1201 in dogs (data not shown). Hence, the strain WH1218 was utilized to conduct subsequent aerosol challenge experiments in mice, dogs and cats.

Table 2 Pathogenicity evaluation of Bb isolates in mice
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Immune response and protective efficacy against Bb challenge in mice vaccinated with different inactivated wild-type strains

Inactivated vaccines containing eight Bb strains with high virulence were injected into mice. Serum samples were collected on days 0, 14, and 28 postimmunization (dpi), and at 28 dpi, the mice were challenged with the wild-type strain WH1218 (Fig. 4A). Bb-specific IgG antibodies in the serum of immunized mice were measured by ELISA. No significant differences in Bb-specific IgG antibody production were detected among the different inactivated Bb strain vaccination groups (Fig. 4B). After challenge with the wild-type strain WH1218, the mice in the PBS group exhibited severe clinical symptoms, such as ocular and nasal discharge, anorexia, depression, intense abdominal respiration, fur dishevelment, and weight loss beginning at 2 days postchallenge (dpc), with a mortality rate of 100% (5/5). Conversely, mice vaccinated with the various inactivated wild strains displayed relatively mild clinical symptoms after challenge, including fur dishevelment and abdominal respiration. Notably, the surviving mice began to recover at 7 dpc. Among the vaccinated groups, mice inoculated with the inactivated vaccine containing the WH1218 strain had the highest survival rate of 80% (4/5). This was followed by the WH1203 and WH1224 strains, both of which exhibited a survival rate of 60% (3/5). The WH1212 and WH1811 strains exhibited a survival rate of 40% (2/5). The WH1816, WH1206, and WH1201 strains had the lowest survival rate of 20% (1/5) (Fig. 4C).

Fig. 4
figure 4

Immunogenicity evaluation of inactivated Bb in mice. A Immunization procedures in mice. B IgG antibody levels against the WH1218 strain on days 0, 14 and 28 after immunization with eight different inactivated strains. C Survival curve of mice after immunization with eight different inactivated Bb strains. Ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001, and ****, p < 0.0001

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Immune response and protective efficacy against Bb challenge in dogs vaccinated with the Bb WH1218, WH1203 and WH1224 strains

The inactivated vaccines containing 3 Bb strains (WH1203, WH1218 and WH1224) with the best protective efficacy in mice were injected into dogs on days 0 and 21. Serum samples were collected on days 0, 7, 14, 21 28 and 35 after primary vaccination, and at 14 days after the second immunization, the dogs were challenged with the wild-type strain WH1218 (Fig. 5A). Bb-specific IgG antibodies in the serum were measured by ELISA. No significant differences in the levels of Bb-specific IgG antibodies were detected among the different groups vaccinated with the wild-type strains (Fig. 5B). Upon challenge, the PBS group exhibited severe clinical symptoms, which peaked at 7 dpc and were characterized by ocular and nasal discharge, sneezing, poor appetite, depression, severe abdominal breathing, frequent spontaneous coughing, and retching, with a morbidity rate of 100% (5/5). In contrast, experimental dogs vaccinated with various inactivated wild-type strains showed mild clinical symptoms after challenge, characterized by ocular and nasal discharge, sneezing and episodic coughing. Furthermore, the average clinical scores of the WH1218, WH1203, and WH1224 strain vaccination groups were significantly lower than those of the PBS group (Fig. 5C). Specifically, the WH1218 inactivated vaccine exhibited the highest protection rate of 100% (5/5), which was 80% (4/5) for the WH1203 strain and 60% (3/5) for the WH1224 strain (Fig. 5D). These results showed that the WH1218 strain is a promising Bb vaccine candidate.

Fig. 5
figure 5

Investigation of the immunogenicity of the Bb WH1218 strain in dogs. A Experimental strategy. B The levels of Bb-specific IgG antibodies against the Bb WH1218 strain in dogs were measured at different time points (days 0, 7, 14, 21, 28, and 35) following the first immunization with three different inactivated strains. C Clinical scores of dogs immunized with three different inactivated strains after the WH1218 challenge. D Protection curve of dogs immunized with three different inactivated strains after the WH1218 challenge. E The levels of Bb-specific IgG antibodies against the Bb WH1218 strain in dogs were measured at different time points (days 0, 7, 14, 21, 28 and 35) following the first immunization with three different doses of WH1218. F Clinical scores of dogs immunized with three different doses of the WH1218 strain measured after the WH1218 challenge. G Protection curve of dogs washed after WH1218 challenge and immunized with three different doses of the WH1218 strain. H Dogs were immunized with three different doses of WH1218, and the Bb load in the lungs was determined 14 days after the WH1218 challenge. I Pathological lesions in the lungs of dogs in different immunization dose groups at 14 days after challenge. Pathological lesions are indicated with arrows. Ns, not significant; *, p < 0.05; **, p < 0.01; ** *, p < 0.001 and ****, and p < 0.0001

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Immune response and protective efficacy against the WH1218 challenge in dogs and cats inoculated with different doses of the WH1218 strain

Inactivated vaccines containing different doses of the WH1218 strain were injected into dogs on days 0 and 21. Serum samples were collected on days 0, 7, 14, 21, 28 and 35 after primary vaccination, and at 14 days after the second immunization, the dogs were challenged with Bb wild-type strain WH1218 (Fig. 5A). The levels of Bb-specific IgG antibodies in the sera of dogs and cats were measured using ELISA. In dogs, from 14 to 35 days after the first immunization, IgG antibody levels were significantly greater in the 109 CFU-immunized group than in the 108 CFU- and 107 CFU-immunized groups, and no significant difference in IgG antibody levels was detected between the 107 CFU- and 108 CFU-immunized groups (Fig. 5E). Dogs in the control group developed severe clinical symptoms after the challenge, including ocular and nasal discharge, sneezing, anorexia, depression, violent abdominal respiration, frequent spontaneous coughing, and retching. The clinical scores peaked at 7 dpc, and the morbidity rate was 100% (5/5). The clinical scores of the immunization groups inoculated with different doses of the WH1218 strain were significantly lower than those of the PBS group (Fig. 5F). The protection rates of the 107 CFU-, 108 CFU- and 109 CFU-immunized groups were 0 (0/5), 60% (3/5) and 100% (5/5), respectively (Fig. 5G). PCR revealed that the nasopharyngeal swabs from the dogs at 7 and 14 dpc were positive for Bb. In addition, at 14 dpc, the canine lung Bb counts of the 109 CFU-immunized group and 108 CFU-immunized group were significantly different from those of the controls, while those of the 107 CFU-immunized group were not different from those of the controls (Fig. 5H). Pathological examination revealed severe macroscopic pathological lesions in the lungs of the dogs in the PBS group, such as hemorrhage and necrosis; the lungs of the dogs in the low-dose immunization group also showed different degrees of lesions. In addition, the alveolar structure was unclear, with more atrophic collapses and marked thickening of the alveolar wall, as indicated by the black arrows. In contrast, no obvious pathological changes were detected in the 109 CFU-immunized group (Fig. 5I).

In cats, the immunization program was the same as that for dogs (Fig. 6A). At 21 to 35 days after the first immunization, the IgG antibody concentration was significantly greater in the 109 CFU-immunized group than in the 108 CFU- and 107 CFU-immunized groups (Fig. 6B). Cats in the control group developed severe clinical symptoms similar to those of dogs after the challenge, mainly spontaneous coughing and retching; the clinical scores peaked at 7 dpc, and the morbidity rate was 100% (5/5). The clinical scores of the immunized groups decreased as the immunization dose increased and were significantly lower than those of the PBS group (Fig. 6C). The protection rates of the 107 CFU-, 108 CFU- and 109 CFU-immunized groups were 40% (2/5), 80% (4/5) and 100% (5/5), respectively (Fig. 6D). Nasopharyngeal swabs from cats at 7 and 14 dpc were positive for Bb according to PCR. In addition, at 14 dpc, the cat lung Bb counts of the 109 CFU-immunized group, 108 CFU-immunized group, and 107 CFU-immunized group were significantly different from those of the controls (Fig. 6E). Pathological examination revealed severe macroscopic pathological lesions, such as hemorrhage and necrosis, in the lungs of the felines in the PBS group; the lungs of the felines in the low-dose immunization group also exhibited different degrees of lesions. In addition, the alveolar structure was unclear, with more atrophic collapses and marked thickening of the alveolar wall, as indicated by the black arrows. In contrast, kittens in the 109 CFU-immunized group showed no obvious pathological changes (Fig. 6F).

Fig. 6
figure 6

Investigation of the immunogenicity of the Bb WH1218 strain in cats. A Experimental strategy. B The levels of Bb-specific IgG antibodies against the Bb WH1218 strain in cats were measured at different time points (days 0, 7, 14, 21, 28 and 35) following the first immunization with three different doses of WH1218. C Clinical scores of cats immunized with three different doses of the WH1218 strain measured after the WH1218 challenge. D Protection curve of WH1218-challenged cats immunized with three different doses of the WH1218 strain. E Cats were immunized with three different doses of WH1218, and the amount of Bb in the lungs was determined 14 days after the WH1218 challenge. F Pathological lesions in the lungs of cats in different immunization dose groups at 14 days after challenge. Pathological lesions are indicated with arrows. Ns, not significant; *, p < 0.05; **, p < 0.01; ** *, p < 0.001 and ****, and p < 0.0001

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Discussion

Between 2021 and 2023, the percentage of Bb-positive dogs in eight provinces in China ranged from 15.47% to 36.23%, and the percentage of Bb-positive dogs with respiratory diseases also confirmed that Bb is one of the main pathogenic bacteria for CIRDC (Bemis et al. 1977a; Maboni et al. 2019). It has been reported in the literature that canine Bb infections are common in the UK, USA, Iran, and Japan and dominate the infectious respiratory disease complex of the CIRD, and an Iranian study reported that cats had a positive rate of 39.13% for pharyngeal samples and 15.78% for nasal samples (Matsuu et al. 2020; Singleton et al. 2019; Tabatabaei and Rohani 2022; Yondo et al. 2023). These results demonstrated that Bb was prevalent in dogs and cats worldwide. In addition, some of the recent reports of Bb infections in China have branched out mainly to economic animals such as pigs and rabbits (Wang et al. 2020; Xu et al. 2023; Zhang et al. 2021). The isolation rate was 1.87% (36/1922) for dogs and cats in China from 2021 to 2023, which is much lower than that reported for pigs with clinical respiratory disease in China from 2018 to 2020 (1.87% vs. 4.25%, P < 0.05) (Zhang et al. 2021). It could be that we have a smaller percentage of samples with respiratory tracts, or it could be that dogs and cats receive more care as companion animals.

VFGs play a pivotal role in the pathogenesis of bacteria (Sharma et al. 2017). These VFGs facilitate the invasion of Bb into its host (Fingermann and Hozbor 2015). Notably, fhaB, prn and bteA exhibited a 100% detection rate; dnt, 91.67%; and cyaA, only 22.22%. Of all the Bb strains analyzed in this study, only isolates from genotype ST27 were not β-hemolytic (data not shown); these strains lost the hemolytic activity of CyaA, which is consistent with previous studies (Buboltz et al. 2008). Since CyaA is antigenic (Gueirard and Guiso 1993), B. bronchiseptica strains lacking this antigen can evade anti-CyaA antibody responses, potentially giving this strain a selective advantage for living in hosts. Another possibility is that strains lacking CyaA may not be as lethal and may allow more hosts to survive and transmit the bacteria (Blaser and Kirschner 2007). These studies demonstrated that most isolates lacked CyaA. However, more than 90% positivity for CyaA has been reported in Chinese pigs and rabbits, possibly because their samples were all from morbid animals (Wang et al. 2020; Zhang et al. 2021), whereas most of the samples in our study were from healthy dogs, resulting in a lower positivity rate of CyaA. In a murine model of infection, CyaA contributes to pathology, lethality, and the colonization and persistence of Bb and B. pertussis in the lower respiratory tract (Guiso et al. 1989; Harvill et al. 1999; Khelef et al. 1992; Weingart and Weiss 2000). Surprisingly, we found that cats were more sensitive to the WH1218 strain challenge, since the challenge dose of dogs was tenfold greater than that of cats, despite this strain being isolated from dogs. This result suggests that cats may be more sensitive to Bb than dogs and complements the absence of cats from previous reports of Bb susceptibility to different species (Bemis 1992).

The administration of antimicrobials is still one of the most effective ways to control Bb and other bacteria, but the emergence of drug-resistant bacteria may lead to the failure of the use of antibiotics in the clinic (Lappin et al. 2017). Monitoring the antimicrobial resistance of clinical microbes is essential for epidemiological studies (Wang et al. 2020). In this study, most of the strains were sensitive to tetracycline (94.44%) and norfloxacin (88.89%), which is consistent with previous reports in pigs and rabbits (Wang et al. 2020; Zhang et al. 2021). This result suggested that these two antibiotics could be effective for treating Bb infections. However, tetracycline should be avoided as much as possible in young animals, with the potential for adverse effects on bones and teeth (Meyers et al. 2020). In addition, most of the isolates were sensitive to amikacin, gentamicin, kanamycin, doxycycline, florfenicol, enrofloxacin, ciprofloxacin, chloromycetin and polymyxin B, suggesting that these antibiotics might be suitable candidates for treating Bb infections when necessary. Cases of bronchial septic bordetellosis treated with nebulized gentamicin have been reported (Clemmons et al. 2021; Morgane Canonne et al. 2020), and the low systemic bioavailability of nebulized gentamicin effectively prevents side effects such as nephrotoxicity and ototoxicity (Al-Amoud et al. 2005). All the isolates were resistant to fosfomycin (100%), followed by ampicillin (91.67%). These findings were consistent with those of previous studies (Kadlec and Schwarz 2018; Wang et al. 2020). Therefore, these drugs are not recommended for use in clinical settings. Our study also revealed that dog-derived strains were generally sensitive to tetracycline and resistant to the compounds neomycin and rifampin, but the opposite results were obtained for cat-derived strains. Doxycycline is regarded as a first-line antibiotic for the treatment of infections caused by Bb (Lappin et al. 2017), possibly because of the overuse of doxycycline in the clinical setting. However, more studies on cat-derived isolates are needed to support our conclusion considering the small number of cat-derived Bb isolates. Our data showed that most Bb isolates in China were multidrug resistant, especially to aminoglycosides, broad-spectrum cephalosporins, and penicillins. This finding is in line with studies of porcine Bb strains in China (Zhang et al. 2021). However, aminoglycoside, broad-spectrum cephalosporin and penicillin antibiotics are commonly used in veterinary medicine for the treatment of Mycobacterium bronchisepticum infections. Therefore, there is a need for continuous clinical monitoring of the prevalence of and trends in these multidrug resistance patterns in isolates (Kadlec and Schwarz 2018; Lappin et al. 2017).

In a series of reports, inactivated parenteral vaccines for Bb failed to provide complete protection against Bb wild-type strain challenge (Bemis et al. 1977b; McCandlish et al. 1978a, b). In our study, dogs and cats received two subcutaneous immunizations of an inactivated vaccine with 109 CFU of WH1218 at three-week intervals, which provided complete protection against Bb challenge. This might be due to differences in the immunogenicity and immunizing dose of the Bb strain. Our results showed that the levels of Bb-specific IgG antibodies in the serum of both cats and dogs inoculated with different doses of the WH1218 strain did not significantly differ after the first vaccination and increased sharply after the second vaccination. In addition, mice that received only a single vaccination could not provide full protection. These results demonstrated that a second or “booster” immunization was necessary for complete protection against Bb infection. Several studies have reported an association between both mucosal and systemic Bb antibody responses and subsequent disease sparing in Bb-infected dogs and other species (Ellis et al. 2001; Goodnow et al. 1979; McCandlish et al. 1978a; Shade and Goodnow 1979; Stephenson et al. 1989). However, we detected serum Bb-specific IgA and nasal lavage Bb-specific IgA and IgG in dogs and cats, which were not significantly different from those in controls (data not shown). Our results show that reliance on a systemic antibody response is sufficient to prevent infection with wild Bb. Th1, Th2 and Th17 responses are reportedly activated in mice after inactivated Bb vaccination (Cui et al. 2022). There are also reports that Th1 and Th17 responses contribute to the clearance of bacteria after Bp infection in mouse models, which further confirms the feasibility of an all-bacteria inactivated vaccine (Borkner et al. 2021; Dubois et al. 2021; Ross et al. 2013). In our study, Bb-specific IgG antibodies were positively correlated with protection rates when dogs were immunized with vaccines containing different concentrations of the WH1218 strain, whereas no correlation was found between Bb-specific IgG antibodies and protection rates when dogs were immunized with vaccines containing different Bb isolates. We hypothesized that Bb-specific IgG antibodies might not be a suitable factor to estimate the protection of different Bb strains, and other factors, such as mucosal immunity and Th1 and Th17 responses, should be explored in immunoprotection experiments (Bey et al. 1981; Chamorro et al. 2023; Yount et al. 2019).

Conclusion

This study provides insights into the prevalence, antibiotic resistance, VFGs, virulence and immunization strategies related to Bb in dogs and cats in parts of China from 2021 to 2023. The results showed that Bb is a crucial pathogen that causes canine respiratory diseases in dogs. Most Bb isolates showed multiple drug resistance traits and possessed at least 4 VFGs. These results highlight the need for ongoing monitoring of the prevalence, VFGs and antibiotic resistance of Bb in China. Notably, we identified an effective inactivated vaccine candidate Bb strain, WH1218, which could provide complete production against Bb wild-type strain challenge in dogs and cats.

Methods

Sample collection

From 2021 to 2023, 1809 canine and 113 feline nasopharyngeal swab samples were collected from eight provinces of China, namely, Guangdong, Henan, Hubei, Shandong, Fujian, Guangxi, Hunan and Liaoning (Fig. 1A), for the isolation and identification of Bb. Among the canine samples, 153 were supplied by veterinarians or collected from pet markets from dogs with respiratory symptoms, and the other 1656 samples were collected from rescue shelters or kennels from dogs with no respiratory symptoms. In addition, 113 feline samples were collected from field cats or pet cats with no respiratory symptoms via a random sampling method.

Bacterial isolation and identification

PCR was used to identify Bb based on the Fla gene locus with the primers listed in Table 3 (Hozbor et al. 1999). PCR-positive swab samples were vortexed with TSB medium containing 5% fresh bovine serum. Then, each swab sample was streaked on a tryptic soy agar (TSA; Becton, Dickinson and Company, MD, USA) plate containing 10 μg/mL nicotinamide adenine dinucleotide (NAD; Sigma, St. Louis, MO) and 10% newborn bovine serum. The agar plates were incubated at 37°C for 24–48 h. The plate-grown isolates were then purified and cultured using standard methods for bacterial identification (Jorgensen et al. 2015). On each agar plate, five colonies with morphological characteristics similar to those of Bb (small, round, shiny, or rough colonies with a diameter of 0.5 to 1.0 mm) were selected and verified via PCR and biochemical evaluation (Woolfrey and Moody 1991).

Table 3 Primers used in the present study
Full size table

Antimicrobial susceptibility evaluation

The disc diffusion method was used to evaluate the antimicrobial susceptibility of the Bb isolates (CLSI 2022). Specifically, overnight-cultured colonies of Bb from TSA plates were purified and suspended in sterile 0.9% normal saline to obtain a 0.5 McFarland standard. The resulting suspension was coated onto Mueller–Hinton (MH) agar (Sigma-Aldrich, 102 St. Louis, MO) using sterile swabs, after which the discs impregnated with specific antibiotics (Hangzhou Microbial Reagent, Hangzhou, China) were placed on agar plates. After five min of drying, the agar plates were incubated overnight at 37°C. The antibiotics used in this study included amikacin (AMK; 30 μg), kanamycin (KAN; 30 μg), doxycycline (DOX; 30 μg), streptomycin (SM; 10 μg), selectrin (SXT; 250 μg), rifampicin (RIF; 5 μg), norfloxacin (NOR; 10 μg), fosfomycin (FOS; 200 μg), ciprofloxacin (CIP; 5 μg), gentamicin (GEN; 10 μg), ceftriaxone (CRO; 30 μg), cefotaxime (CTX; 30 μg), enrofloxacin (ENR; 10 μg), chloramphenicol (CHL; 30 μg), florfenicol (FLO; 30 μg), ampicillin (AMP; 10 μg), tetracycline (TET; 30 μg), and polymyxin B (PMB; 300 IU). After incubation, the bacterial inhibition zone diameter was measured, and the results were interpreted according to the Clinical and Laboratory Standards Institute document (CLSI 2022). Owing to the limited availability of clinical breakpoints specific to Bb (Kadlec and Schwarz 2018), we employed breakpoints of Enterobacteriaceae in the CLSI document M100 to interpret our results. In addition, we utilized Escherichia coli ATCC®∗25922 for quality control.

Detection of virulence factor-encoding genes (VFGs)

The presence of five well-characterized VFGs of Bb isolates, including the filamentous hemagglutinin-encoding gene fhaB, the pertactin-encoding gene prn, the adenylate cyclase-hemolysin toxin-encoding gene cyaA, the dermonecrotic toxin-encoding gene dnt, and the Bordetella type-III secretion system effector A-encoding gene bteA, was examined by PCR with the primers listed in Table 3. The 20 μL PCR system contained 2 μL of bacterial DNA, 1 μL of each forward and reverse primer, 10 μL of 2 × Taq Master Mix (Dye Plus), 2 μL of DMSO, and 4 μL of ultrapure water. PCR procedures were as follows: an initial phase at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 5 min. Bb strain HH0809 (Ai et al. 2019), preserved in the laboratory of Huazhong Agricultural University, was used as a positive control, and sterile ultrapure water was used as a negative control. Finally, the PCR products were analyzed by electrophoresis on a 1% agarose gel.

Virulence and protective efficacy of Bb isolates in mice

To determine the virulence of the isolated Bb strains, 55 4-week-old female BALB/c mice were divided into 11 groups with 5 mice per group, and Groups 1–10 were challenged intraperitoneally with one of 10 Bb strains isolated from the dogs with respiratory symptoms (0.2 mL/mouse, 107 CFU/mouse). The same volume of normal saline (0.2 mL/mouse) was injected into Group 11 as a control. After challenge, the clinical symptoms of the mice were observed for 14 d, and the mice were euthanized when the weight decreased to 75% of their original body weight. At 14 d postchallenge (dpc), all the mice were euthanized.

To determine the protective efficacy of Bb strains in mice, 8 Bb strains with high virulence were adjusted to 109 CFU/mL, inactivated by beta-propiolactone (1:1000) and mixed with aluminum glue adjuvant at a final concentration of 0.5 mg/mL to obtain vaccines. Four-week-old 90 BALB/c mice were divided into nine groups with 10 mice per group. Groups 1-8 were injected intramuscularly with the inactivated vaccines, and Group 9 was injected with the same amount of PBS as a negative control (Fig. 4A). On 0, 14, and 28 dpi, serum samples were collected from the tail vein of the mice to track their Bb-specific IgG antibodies. At 28 dpi, the mice were challenged with Bb wild-type strain WH1218 (method 2.8). After challenge, the clinical symptoms of the mice were observed for 14 days, and the mice were euthanized when their body weight decreased to 75%.

Immune responses and protective efficacy after immunization with WH1203, WH1218 and WH1224 in dogs

To evaluate the immune responses and protective efficacy of Bb isolates in dogs, three isolates (WH1203, WH1218 and WH1224) with the best protective efficacy in mice were adjusted to 109 CFU/mL, inactivated by beta-propiolactone (1:1000), and mixed with a water adjuvant to obtain an inactivated vaccine. Twenty healthy 6- to 10-week-old dogs free of antigens and antibodies to Bb were randomly allocated to one of four groups (n = 5 dogs/group). Groups 1-3 were injected subcutaneously with 1 mL (109 CFU/mL) of the inactivated vaccine containing the WH1203 strain, the WH1218 strain or WH1224 on days 0 and 21, and group 4 was injected with the same amount of PBS as a control (Fig. 5A). Serum samples were collected on days 0, 7, 14, 21, 28 and 35 after primary vaccination to track their Bb-specific IgG antibodies. At 14 d after the second immunization, all the canines were challenged with the Bb wild-type strain WH1218. After challenge, clinical scores (according to Table 4) (Ellis et al. 2016) were recorded daily for 14 days, and the dogs were euthanized when their scores exceeded 12 points. At 14 dpc, all dogs were euthanized.

Table 4 Clinical scoring rubric (Ellis et al. 2016)
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Immune responses and protective efficacy after immunization with different doses of the WH1218 strain in dogs and cats

To further evaluate the immune responses and protective efficacy of the selected strain WH1218 in dogs and cats, the WH1218 strain was inactivated by beta-propiolactone (1:1000) and mixed with a water adjuvant to obtain an inactivated vaccine containing the WH1218 strain at final concentrations of 107 CFU/mL, 108 CFU/mL, or 109 CFU/mL. Twenty healthy 6- to 10-week-old dogs or cats free of antigens and antibodies to Bb were randomly allocated to one of four groups (n = 5 dogs each). The dogs and cats in groups 1-3 were injected subcutaneously with 1 mL of the inactivated vaccine containing 107 CFU/mL, 108 CFU/mL or 109 CFU/mL of the WH1218 strain on days 0 and 21, and group 4 was injected with the same amount of PBS as a control (Fig. 5A). Serum samples were collected on days 0, 7, 14, 21, 28 and 35 after primary vaccination to track their Bb-specific IgG antibodies. At 14 days after the second immunization, all the dogs or cats were challenged with the Bb wild-type strain WH1218. After the challenge, clinical scores (according to Table 4) (Ellis et al. 2016) were recorded daily for 14 days, and dogs or cats were euthanized when their scores exceeded 12 points. At 14 dpc, all dogs or cats were euthanized, and lung samples were collected for analysis of pathological lesions and Bb counts.

Mouse, dog and cat challenge experiments

The highly virulent Bb WH1218 strain was cultured on selective Bordet Gengou (BG) agar plates following previously described procedures (Ellis et al. 2001). The relationship between colony forming units (CFUs) and turbidity during the culture process was predetermined, and the turbidity of the culture suspension was measured to determine the amount of live WH1218 strain in the culture suspension before challenge. Ten mice were exposed to 12 mL of an atomized culture suspension containing approximately 1.2 × 1013 CFU/mL (1 × 106 CFU per mouse) of the WH1218 strain for 45 min in a confined space (35 cm in length, 20 cm in width, 20 cm in height). At one time, 5 dogs or cats were exposed to 8 ml of atomized culture suspension containing approximately 8 × 1010 CFU for dogs (1 × 1010 per dog) or 8 × 109 for cats (1 × 109 per cat) for 30 min in a confined space (60 cm in length, 50 cm in width, and 50 cm in height).

Measurement of Bb-specific IgG and IgA by ELISA

Bb-specific IgG and IgA antibodies were measured by ELISA as previously described (Boot et al. 1993). Briefly, the culture suspension of the WH1218 strain was centrifuged, and the collected bacteria were washed with PBS 3 times. The bacteria were subsequently sonicated and resuspended in physiological saline and stored at -70°C until use. Ninety-six-well flat-bottomed microtiter plates were coated with 7.5 μg of antigen per well dissolved in carbonate buffer and incubated overnight at 4°C. After removing the antigen coating, the plate was washed with double-distilled water containing 0.05% Tween 20 and then incubated with 1% bovine serum albumin (BSA) at 37°C for 2 h. Serum samples were diluted in PBS at a 1:50 ratio and added to the plate at 50 μL per well. The plate was incubated at 37°C for 1 h, washed, and incubated with horseradish peroxidase (HRP)-labeled rabbit anti-cat IgG (1:10000), rabbit anti-dog IgG (1:10000), goat anti-mouse IgG (1:10000), and goat anti-mouse IgA (1:10000) at 37°C for 60 min. The results were read at 630 nm. Sera from species that were neither vaccinated nor challenged with Bordetella were used as negative controls, and those from species that were vaccinated or challenged with Bordetella were used as positive controls. Blank wells contained a PBS solution with 0.05% Tween 20 and 0.2% gelatin.

Clinical scoring and morbidity criteria for dogs and cats

Dogs and cats were clinically scored according to a previously reported scale involving the assessment of nasal discharge, eye droppings, coughing, sneezing, depression, regurgitation, and respiration, with the severity of each symptom ranging from 0 to 3 points (Table 4) (Ellis et al. 2016). The morbidity criteria were when dogs or cats coughed or their clinical scores were greater than 3 for two consecutive days.

Statistical analysis

Statistical analysis was conducted using one-way ANOVA or Student’s t test in GraphPad Prism V. 8.3.0. The data are expressed as the mean ± standard deviation (SD). The levels of significance were set as follows: P < 0.05 (*) denoting a significant difference, P < 0.01 (**) indicating a highly significant difference, and P < 0.001 (***) representing an extremely significant difference.

Availability of data and materials

The data that support the findings of this study are openly available in this manuscript and are available from the corresponding author upon request.

Abbreviations

Bb :

Bordetella bronchiseptica

VFGs:

Virulence factor encoding genes

CIRDC:

Canine infectious respiratory disease

CFU:

Colony forming unit

MR:

The methyl red

VP:

Voges-Proskauer

dpi:

Days postimmunization

dpc:

Days postchallenge

CLSI:

Clinical and Laboratory Standards Institute

fhaB :

Filamentous hemagglutinin-encoding gene

prn :

Pertactin-encoding gene

cyaA :

Adenylate cyclase-hemolysin toxin-encoding gene

dnt :

Dermonecrotic toxin-encoding gene

bteA :

The Bordetella type-III secretion system effector A-encoding gene

References

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Acknowledgements

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Funding

This study was supported by the Guangdong Major Project of Basic and Applied Basic Research (2020B0301030007).

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Authors and Affiliations

  1. National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, China

    Qinghua Shang, Wenhui Gao, Xiangting Zhang, Jianqing Zhao, Ying Wu, Haoqi Li, Zhen F. Fu, Chengguang Zhang & Ling Zhao

  2. Key Laboratory of Preventive Veterinary Medicine of Hubei Province, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China

    Qinghua Shang, Wenhui Gao, Xiangting Zhang, Jianqing Zhao, Ying Wu, Haoqi Li, Ming Zhou, Zhen F. Fu, Chengguang Zhang & Ling Zhao

  3. Hubei Hongshan Laboratory, Wuhan, 430070, China

    Ling Zhao

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  1. Qinghua Shang
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Contributions

QS, LZ, and CZ delineated the study conception and design. LZ and CZ supervised the study. QS, WG, XZ, HL, YW, and JZ collected the bacterial isolates, performed laboratory tests and analyzed the data, and QS, WG and XZ participated in the animal experiments and analyzed the data. QS, CZ and JZ wrote the manuscript and approved the final version for publication. CZ, LZ, MZ, ZF, and HC participated in the manuscript discussion and revision. All authors have read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Chengguang Zhang or Ling Zhao.

Ethics declarations

Ethics approval and consent to participate

All mouse studies were conducted at the Laboratory Animal Center of Huazhong Agricultural University, and the mice were fed in specific pathogen-free (SPF) environments. During the Bb challenge study, the mice with a body weight decrease of more than 25%, severe paralysis, or inability to feed were euthanized by CO2. The experimental protocol was reviewed and approved by the Scientific Ethics Committee of Huazhong Agricultural University with the approval number HZAUMO-2020–0096. The dogs and cats used in this study were fed in isolation environments as recommended by the Regulations for the Administration of Affairs Concerning Experimental Animals of the Ministry of Science and Technology of China. The immunization and challenge of Bb experiments were performed using protocols that were approved by the Scientific Ethics Committee of Huazhong Agricultural University (permit numbers: HZAUCA-2020-002 and HZAUDO-2020–003).

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Our research paper agreed to be published in Animal Diseases.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study. Author Ling Zhao was not involved in the journal’s review or decisions related to this manuscript.

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Shang, Q., Gao, W., Zhang, X. et al. Investigation of the epidemiology, pathogenicity and immunogenicity of Bordetella bronchiseptica isolated from cats and dogs in China from 2021 to 2023. Animal Diseases 4, 16 (2024). https://doi.org/10.1186/s44149-024-00120-3

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Keywords

Animal Diseases

ISSN: 2731-0442