First Isolation of Brucella canis from Pet Dogs in Sichuan Province, China: Molecular Characterization, Pathogenicity and Antigen Location Analysis

Zhijun Zhong1, Rui Tu1, Xichun Wang2, Yi Geng1, Qicheng Xiao1, Yinan Tian1, Bin Wei1, Jiaming Dan1, Ya Wang1 and Guangneng Peng1,*

1Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China

2College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, P.R. China

Zhijun Zhong and Rui Tu contributed equally to this work.

ABSTRACT

Brucellosis is a worldwide zoonosis that is primarily caused by Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis. However, in China, information regarding brucellosis in pet dogs caused by B. canis is limited. In the present study, we conducted a comprehensive molecular, pathological, and immunohistochemical analysis to detect this pathogen in pet dogs. Molecular methods, combining three types of PCR assays, identified two strains isolated from two pet dogs as Brucella canis. Histopathological changes revealed extensive inflammation and necrosis in the liver, lung, spleen, kidney, testicle, and lymph nodes, among which changes in the spleen were the most serious. Immunohistochemistry results demonstrated the detection of B. canis antigens in the lesions of all examined tissues. Strong positive staining was primarily found in the spleen, liver, and testicle. In conclusion, this study was the first to report the isolation of two B. canis strains from pet dogs in Sichuan province, southwestern China, and to further evaluate B. canis antigen location in tissues. Our study will contribute to the understanding of B. canis pathogenicity in naturally-infected pet dogs.

Article Information

Received 21 December 2017

Revised 11 March 2018

Accepted 4 April 2018

Available online 31 October 2019

Authors’ Contribution

YG, YW and QX collected the samples and analyzed the data. ZZ, RT and XW wrote the article. ZZ, YT, RT, JD and BW performed the experiment. ZZ and GP helped in preparation of the article.

Key words

Brucella canis, Pet dog, Molecular diagnostics, Pathology, Immunohistochemistry.

DOI: https://dx.doi.org/10.17582/journal.pjz/2020.52.1.283.291

* Corresponding author: [email protected]

0030-9923/2020/0001-0283 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

Introduction

Brucellosis, caused by gram negative and aerobic, facultative bacteria of the genus Brucella, is regarded as one of the most important zoonotic diseases worldwide (Zhong et al., 2013). In recent years, an increasing number of reports have described brucellosis caused by Brucella canis, suggesting that this disease might be increasing in incidence (Agudelo-Flórez et al., 2012; Gyuranecz et al., 2011b; Hofer et al., 2012; Holst et al., 2012; Kang et al., 2011; Keid et al., 2017; Kulakov, 2012; Purvis et al., 2017; Sayan et al., 2011); however, B. canis infection in dogs has not conventionally been considered a major problem. This species was first isolated from dogs in 1966 and has since been considered a threat to animal and human health (Krueger et al., 2015; Makloski, 2011). To date, dogs infected with B. canis has been primarily reported in European countries such as Sweden, Italy, Germany, Poland, Great Britain, France, Spain, Russia, Hungary, Austria; in addition, it is also prevalent in Asian countries such as Korea, Japan, China, Turkey, India, the Philippines, and Malaysia, as well as in the Americas including the United States, Canada, Brazil and Columbia (Brennan et al., 2008; Holst et al., 2012; Keid et al., 2017; Purvis et al., 2017). However, information regarding B. canis infection in pet dogs in China is scant.

In China, B. canis was first isolated from Beagle dogs in Shanghai (Deqiu et al., 2002). Subsequently, more strains were isolated in different provinces such as Guangxi, Henan, Inner Mongolia, Beijing, Guangxi, Anhui, Hubei, Jiangsu, Xinjiang, Shanxi, and Fujian, indicating that the prevalence of B. canis infection in dogs is increasing in China (Deqiu et al., 2002). This species is not only highly pathogenic for dogs, but it is also able to infect humans and eventually cause severe diseases (Krueger et al., 2015; Lucero et al., 2005; Marzetti et al., 2013). Humans become infected through close contact with infected dogs or abortion-related materials (Lucero et al., 2010). In the last decade, there has been a rising trend of human brucellosis caused by B. canis (Lucero et al., 2010; Nomura et al., 2010). Sichuan province is the second most populated province in China and contains a large amount of pet dogs. Therefore, infected pet dogs could become a major threat to dog owners. However, little information is available regarding B. canis infection of pet dogs in Sichuan province. Considering the high-density feeding environment and the increase in reports of B. canis infection in pet dogs in recent years, canine brucellosis might become an emerging challenge to animals and public health in China.

In this study, we combined the advantage of AMOS-PCR and BcSS-PCR to distinguish B. canis from other Brucella species. Then, pathologic and immunohistochemical (IHC) techniques were employed to detect pathological changes and B. canis antigens in tissues from a naturally-infected dog. This is the first study reporting the isolation of B. canis strains from pet dogs, in addition to the use of IHC methods to detect B. canis antigen locations in a naturally-infected pet dog, in Sichuan province, China.

 

Materials and methods

Clinical samples

In December 2015, one male dog (golden retriever), aged two years and five months, presented with low fever, enlarged lymph nodes, and unilateral testis, indicating the possibility of canine brucellosis. In October 2016, another dog, a male poodle, aged three years, presented with obviously enlarged testicle and undulating fever, and was sent to Veterinary Medical Teaching Hospital of Sichuan Agricultural University.

Serological tests

The rapid slide agglutination test (RSAT) and Rose Bengal plate test (RBPT) were performed to detect Brucella antibodies in blood samples, and were performed as previously reported (Ali et al., 2017). For further detection of rough Brucella antibodies, we adopted the Rose Bengal plate test (RBAT) with rough antigen.

Bacteriological studies

Blood samples were plated on tryptic soy agar (Beijing Selarbio Science and Technology Co., Ltd, Beijing, China) and streaked for isolation. Plates were inoculated at 37 °C in 5% CO2 for greater than 5 days. Smooth or rough colony phenotypes of isolates were confirmed by crystal violet staining and auto agglutination reactions as described previously (Alton et al., 1998). Additional bacteriological identification studies were performed by assessing agglutination with monospecific sera against A and M antigens, hydrolysis of urea, H2S production, and growth in the presence of CO2. The strains mentioned in this study included three Brucella reference strains (544A, 16M, and S19) and the two isolates (W5 and Y4).

DNA extraction

Before DNA extraction, specimens were boiled for 15 min. Brucella DNA was extracted according to the manufacturer’s instructions using, TIANamp Bacteria DNA extraction kit (TIANGEN Biotech Corporation, Beijing, China).

Polymerase chain reaction assays

Three different PCR protocols were used to identify the two Brucella isolates. All primers are listed in Table I. The products (6 µl from each reaction mixture) were analyzed by electrophoresis using a 1.5% agarose gel, after which the1 gel was stained with ethidium bromide and photographed.

Brucella genus-specific PCR

DNA from all isolates was amplified using BCSP31 gene, producing a 224-bp amplicon. Primers used are listed in Table I, and were previously described by Imaoka et al. (2007). Amplification conditions consisted of an initial denaturation at 99 °C for 5 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 7 min.

 

Table I.- Primers used in this study.

Primer	Sequence (5’-3’)
BCSP31	F: TGG-CTC-GGT-TGC-CAA-TAT-CAA
	R: CGC-GCT-TGC-CTT-TCA-GGT-CTG
BcSS	F: CCA-GAT-AGA-CCT-CTC-TGG-A
	R: TGG-CCT-TTT-CTG-ATC-TGT-TCT-T
Specific primer
IS711	TGC-CGA-TCA-CTT-AAG-GGC-CTT-CAT
B. abortus	GAC-GAA-CGG-AAT-TTT-TCC-AAT-CCC
B. melitensis	AAA-TCG-CGT-CCT-TGC-TGG-TCT-GA
B. ovis	CGG-GTT-CTG-GCA-CCA-TCG-TCG
B. suis	GCG-CGG-TTT-TCT-GAA-GGT-TCA-GG

 

AMOS-PCR

The Brucella PCR diagnostic assay primer cocktail was composed of the five primers listed in Table I (Bricker and Halling, 1994). The reaction mixture consisted of 12.5 µl Taq PCR MasterMix (0.1U Taq Polymerase/µl, 500 µM dNTP each, 20 mM Tris-HCl, 100 mM KCl, 3 mM MgCl2), 2 µl of template DNA, 7.9 µl ddH2O, and the five-primer cocktail (0.4 µl B. abortus-, B. melitensis-, B. suis-, B. ovis-, and 1 µl of IS711-specific primer). The PCR conditions consisted of an initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 94 °C for 50 s, annealing at 55 °C for 50 s, and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min (Chen et al., 2006).

BcSS-PCR

B. canis was detected by BcSS-PCR amplification of a 300-bp nucleotide fragment of the BCAN gene. The PCR amplification primers and conditions were previously described by Kang et al. (2014). PCR reactions were performed using a 20-µl reaction mixture containing 10 µl Taq PCR MasterMix, 2 µl of template DNA, 2 µl of each of the primers (10 pmol), and 4 µl ddH2O.

Histopathology and immunohistochemistry

Based on serological tests, the two dogs (golden retriever and poodle) were diagnosed with canine brucellosis. Due to the cost of treatment, the owner of one dog (golden retriever) opted against treatment and signed a consent form for euthanasia and post-mortem examination, according to the recommendations of the Sichuan Agricultural University ethics committee. Humane euthanasia was performed and tissue samples (liver, spleen, kidney, lung, lymph node, and testicle) were removed immediately. Tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned into 5-μm thick sections, and stained with hematoxylin and eosin (HE) following standard procedures (Gyuranecz et al., 2011a). These tissues were further analyzed by IHC analysis following the method previously described by Saglam et al. (2008). The primary antibody was a polyclonal goat antiserum (anti-Brucella Positive Control Serum, China Institute of Veterinary Drug Control, China) at a dilution of 1:50. For various medical reasons, the other dog (poodle) was sent to a different hospital for further treatment, and thus, we could not obtain further treatment information.

 

Results

Serological and bacteriological tests

RSAT and RBPT were all showed positive for the two pet dog’s blood samples. Blood samples from two pet dogs (golden retriever and poodle) yielded colonies that were culturally confirmed to be Brucella. Tests for urease, hydrogen sulfide, and reactions against monospecific sera A and M indicated that the two isolates (W5 and Y4) were B. canis. Bacteriological results are summarized in Table II.

Molecular diagnostics

Three types of PCR assays identified the two strains isolated from two pet dogs as Brucella canis (Table III).

 

Table II.- Results of bacteriological findings for the two isolates from dogs.

Isol-ates	Colony morph-ology	CO2 requir-ement	H2S produ-ction	Hydro-lysis of urea	Agglutination in sera		Auto agglutin-ation with acraflavin	Crystal violet staining
					A	M
W5	Rough	-	-	+	-	-	+	+
Y4	Rough	-	-	+	-	-	+	+

 

Table III.- Results of three PCR assays for amplification of Brucella isolated from five strains in this study.

Species	Strains	PCR results
		Brucella genus specific PCR	AMOS-PCR	BcSS-PCR*
B. abortus	544A	+	+	-
B. melitensis	16M	+	+	-
B. suis	S2	+	+	-
B. canis	W5	+	-	+
B. canis	Y4	+	-	+

*B. canis species-specific PCR performed in this study; + indicates application by PCR; - indicates no application by PCR.

 

Brucella genus-specific PCR, using the five strains (two isolates and three reference strains), successfully produced a 224-bp PCR amplicon (Fig. 1), indicating that all tested strains belong to the Brucella genus. Upon performing AMOS PCR, DNA from isolates Y4 and W5 was not amplify (Fig. 2), indicating that the two isolates do not belong to B. abortus biovar 1, 2, or 4, B. melitensis, B. ovis, or B. suis. BcSS-PCR assays using the two isolates (Y4 and W5) resulted in a specific 300-bp amplicon (Fig. 3), suggesting that the isolates were B. canis.

 

 

Histopathology

Extensive inflammatory and necrotic lesions in the liver, spleen, testicle, lymph node, lung, and kidney were observed. Histopathological changes in the liver were mainly located in the portal tracts, where severe infiltration of abundant lymphocytes and neutrophils was detected. In addition, hepatocellular necrosis and extensive vacuole degeneration in hepatocytes were also observed (Fig. 4A). Microscopic examination of the spleen showed the presence of many granulomas with central necrotic areas in red pulp. Necrotic foci were encircled by several different types of cells, consisting of a large number of necrotic neutrophils, a few plasma cells, and occasional macrophages (Fig. 4B). The principle lesions mainly found in the testicle were severe necrosis of spermatogenic cells and damaged seminiferous tubule structures accompanied by massive neutrophil, lymphocyte, and macrophage accumulation in interstitial tissue (Fig. 4C), resulting in orchitis or epididymitis. The principle alterations observed in the lymph nodes were proliferation of lymphocytes and reticuloendothelial cells along with deposition of fibrinous material. In addition, a bacterial bluish discoloration was found adjacent to the necrotic foci (Fig. 4D).

 

However, changes in the lung and kidney were much milder compared to those of the other four tissues. The main lesions in the lung were low-to-moderate alveolar septum and mesenchyme infiltration by lymphocytes. Changes in the kidney were necrosis and exfoliation of renal tubular epithelial cells accompanied by focal infiltration of inflammatory cells.

Immunohistochemistry

IHC staining showed bacterial antigens in the lesions of various organs. B. canis antigens were primarily located in the cytoplasm of macrophages and neutrophils in portal infiltrates of the liver (Fig. 5A). Brucella antigens were also detected in the cytoplasm of macrophages in the red splenic pulp (Fig. 5B), cytoplasm of epithelial cells of cortical and medullar tubules, and macrophages and neutrophils of the renal interstitium. The cytoplasm of spermatogenic cells, macrophages and neutrophils of the testicle (Fig. 5C), and macrophages of the lymph nodes (Fig. 5D) were all positive for B. canis staining. Positive staining was also found in cellular debris from the alveolar septum of the lung.

 

Discussion

Canine brucellosis is one of the most serious zoonotic diseases, and not only endangers dogs but is also a threat to public health. Dogs can be infected by four Brucella species including B. canis, B. abortus, B. melitensis and B. suis (Hollett, 2006). Among the four species, B. canis is the predominant pathogen causing canine brucellosis (Gyuranecz et al., 2011b). Since the first isolation of B. canis strains from dogs in China, nationwide research regarding the infection caused by B. canis has been carried out in 25 provinces, and more than 300 strains have been isolated in 20 provinces with the infection rate varying from 0.3% to 42.7% (Deqiu et al., 2002). Epidemic regions are mostly located in the north, south, and southeast of China, for example Beijing, Shanghai, Tianjin, and Inner Mongolia (Di et al., 2014). In 2013, a study conducted in Inner Mongolia indicated that the B. canis infection rate in dogs reached 38.37% (Gao, 2013). In 2015, studies in Beijing suggested that this incidence is on an upward trend (Di et al., 2014). Published information suggests that canine brucellosis is more prevalent in China. However, no report is available regarding pet dogs infected with B. canis in Sichuan province, southwest China. In this study, we first isolated this species from two naturally-infected pet dogs in Sichuan, China. Because of the rapid growth of pet and companion animal industries in this region, B. canis infections pose a substantial risk to individuals and public health.

In the present study, the two strains were confirmed by biochemical and molecular methods. It is noteworthy that, a BcSS-PCR assay was employed to identify the two B. canis isolates. This assay was established by Kang et al. (2014)and consists of B. canis species-specific amplification of a 300-bp fragment of the BCAN gene. Therefore, BcSS-PCR should be strongly recommended for the rapid detection of B. canis stains. In addition, we further conducted pathologic and IHC techniques to identify pathology changes and antigen locations in an infected dog (a male golden retriever). Although no obvious clinical signs other than enlarged lymph nodes and unilateral testis were found, pathological examination (in our study) revealed mild-to-severe inflammatory and necrotic lesions in all tested tissues, among which lesions in the liver, kidney, and lymph nodes were similar to those of published reports (Brennan et al., 2008; Carmichael and Kenney, 1970; Gleiser et al., 1971; Gyuranecz et al., 2011b). Previous reports demonstrated that pathologic differences between male and female dogs are remarkable in the spleen except for their own reproductive system (Brennan et al., 2008). In this study, significant necrotic changes were observed in the splenic red pulp with few hyperplastic lesions being observed in white pulp, which corresponds to the findings of Brennan et al. (2008). According to that report, hyperplasia of the splenic white pulp is prominent in females and not in males (Brennan et al., 2008). In addition to sex, there are also differences in the lungs between adult dogs and aborted fetuses. Our study showed that changes in the lung were much less prominent, in agreement with findings described for adult dogs (Brennan et al., 2008; Gleiser et al., 1971; Gyuranecz et al., 2011b). Accordingly, previous studies showed that histological alterations in the lung are the most significant lesions in aborted fetus (Brennan et al., 2008; Hofer et al., 2012; Xavier et al., 2009). The reason for this might be that fetuses are in close contact with vaginal discharges, abortion materials, and fluids with high bacterial loads. IHC techniques have been widely used for the detection of B. abortus, B. suis, and B. melitensis antigens in many animals such as cows, sheep, goats, bovine and ovine aborted fetuses, and hares (Gyuranecz et al., 2011a; Osburn and Kennedy, 1966; Saglam et al., 2008; Xavier et al., 2009). However, information regarding the detection of B. canis antigens in naturally-infected pet dogs is scant, especially in China. In our study, immunolabeling of B. canis antigens was stronger in the spleen, testicle, and liver than in the kidney and lymph nodes, and this was associated with the severity of inflammatory and necrotic lesions in those tissues. The detection is characterized by the observations on histopathology and IHC from a single study case. Yet to get more insight of B. canis pathogenicity in naturally-infected pet dogs, it is important to include more study cases in the future.

Canine brucellosis is an increasing zoonotic risk, with B. canis infections in pet dogs showing an upward trend in recent years (Holst et al., 2012). Given that therapy is not completely effective and the serious zoonotic implications of these infections, euthanasia of infected dogs should be advocated by professional agencies. If treatment must be adopted, infected dogs should be sterilized first. However, the treatment fee is relatively high and the period is lengthy. Additionally, disease relapse is still possible after ending therapy (Makloski, 2011).

 

Conclusion

We reported the first isolation of B. canis strains from pet dogs in Sichuan province, with an analysis of pathogenicity and antigen locations in a naturally-infected animal. This will benefit the epidemiologic, pathologic, and immunohistochemical study of B. canis infection in pet dogs. Future studies are needed to fully elucidate the epidemiology of such infections in China. In addition, the lack of a canine Brucella vaccine might imply a significant threat to animal and public health, which necessitates the future development of a safe and efficient vaccine against B. canis.

 

Acknowledgements

The present study was financially supported by the National Key Research and Development Program of China (2016YFD0501009) and the Chengdu Giant Panda Breeding Research Foundation (CPF2015-4).

 

Ethics statement

This study was reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University under permit number DYY-S20157034. Prior to the collection of specimens from dogs, permission was obtained from owners.

 

Statement of conflict of interest

The authors declare that they have no competing interests.

 

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