Biologicals 42 (2014) 109e113
Contents lists available at ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Prevalence of Met-203 type spaA variant in Erysipelothrix rhusiopathiae isolates and the efficacy of swine erysipelas vaccines in Japan Mariko Uchiyama a, Kinya Yamamoto b, Mariko Ochiai a, Tsukasa Yamamoto a, Fumiya Hirano a, Saiki Imamura a, Hidetaka Nagai a, Kouji Ohishi a, Noriyuki Horiuchi c, Mayumi Kijima a, * a
National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, 1-15-1 Tokura, Kokubunji, Tokyo 185-8511, Japan Animal Products Safety Division, Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, 1-2-1 Kasumigaseki, Chiyoda-ku, Tokyo 100-8950, Japan c Department of Basic Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido 080-8555, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 June 2013 Received in revised form 5 November 2013 Accepted 2 December 2013
Since 2009, erysipelas infection among pigs in Japan has been increasing. This study investigated the prevalence, and characteristics of Erysipelothrix rhusiopathiae isolates in Japan from 2008 to 2010 and assessed the efficacy of current commercial erysipelas vaccines. Based on polymorphisms in a 432-bp hypervariable region in the surface protective antigen A (spaA) gene, 34 isolates were classified into three groups: (i) Group 1 with methionine at position 203 (Met-203) and isoleucine at position 257 (Ile257) (18 isolates of serotype 1a and one untypable isolate). (ii) Group 2 with Ile-257 (12 isolates of serotypes 1a, 1b, 2, 10 and 11), and (iii) Group 3 with alanine at position 195 (Ala-195) and Ile-257 (three isolates of serotype 1a). Isolates with Met-203 were highly pathogenic in mice and pigs, causing death in the pig and LD50 values of 0.45e1.45 CFU per mouse. One live and three inactivated commercial E. rhusiopathiae vaccines were evaluated for efficacy against a Met-203 isolate. Almost all mice and pigs that received vaccine survived, while non-vaccinated controls all died within 5 days of the challenge. This indicates that swine erysipelas vaccines might be still effective in protecting animals against the recently prevalent Met-203 isolates in Japan. Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Erysipelothrix rhusiopathiae Vaccine spaA
1. Introduction Erysipelothrix rhusiopathiae is a Gram-positive bacillus that causes erysipelas in a variety of mammals and birds, and dermatological erysipeloid disease in humans [1,2]. Erysipelas is commonly associated with swine, and can be divided into three types by its clinical signs: acute (septicemia), sub-acute (urticaria), and chronic (arthritis, lymphadenitis, and endocarditis). Acute swine erysipelas is characterized by generalized septicemia often resulting in sudden death, and is specified as a notifiable infectious disease in Japan. Pigs infected with erysipelas are prohibited from being slaughtered or are condemned at the slaughterhouse by the Slaughterhouse Act, to prevent human consumption. The pig industry therefore suffers considerable economic loss from this disease. * Corresponding author. Tel.: þ81 42 321 1841; fax: þ81 42 321 1769. E-mail address: [email protected] (M. Kijima).
For the prevention of swine erysipelas, attenuated live vaccine prepared from the acriflavine-fast attenuated Koganei strain 65e 0.15 of E. rhusiopathiae has been used in Japan for many years because of its low-cost and convenience. Appropriate inoculation with the live vaccine produces a strong immunity [3,4]. An inactivated vaccine was approved in 1997 because the live vaccine had a higher incidence of adverse events in pigs kept in a clean environment, such as specific-pathogen-free (SPF) pigs [5,6]. Several kinds of inactivated vaccines have been approved in Japan for swine erysipelas, and their use has been increasing in recent years. The surface protective antigen (Spa) protein of E. rhusiopathiae has been shown to be highly immunogenic and is a promising vaccine candidate for erysipelas [7,8]. The Spa proteins of E. rhusiopathiae can be classified into 3 molecular species, named SpaA (including serotypes 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17 and N), SpaB (serotypes 4, 6, 11, 19 and 21) and SpaC (serotype 18) [8]. A mouse cross-protection study showed that three recombinant Spa proteins (rSpaA, rSpaB and rSpaC) elicited complete protection against
1045-1056/$36.00 Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biologicals.2013.12.002
110
M. Uchiyama et al. / Biologicals 42 (2014) 109e113
challenge with homologous strains but that the level of protection against heterologous strains varied depending on the rSpa protein used for immunization [8]. This indicated that the Spa proteins, especially the N-terminal half of the hyper-variable region, are important for specific immunity. A recent report by To et al. [9] demonstrated that a variant isolate with aspartic acid (Asp) and methionine (Met) at the amino acid positions 195 (Asp-195) and 203 (Met-203), respectively, in the hyper-variable region of the spaA gene might be widespread among pig populations in Japan. The Met-203 isolates, therefore, differ in nucleotide sequence, in the 432-bp hypervariable region of the spaA gene, from the live vaccine strain Koganei 65-0.15 [9]. Unfortunately, according to national statistics, the morbidity rate of swine erysipelas in Japan has been increasing since 2009 [10]. The aim of this study was to characterize recently isolated E. rhusiopathiae strains, assess the prevalence and pathogenicity of the spaA variants, and re-evaluate the validity of swine erysipelas vaccines against a spaA variant field isolate. 2. Materials and methods 2.1. Bacterial strains and growth conditions A total of 34 E. rhusiopathiae isolates were collected for this study. They were isolated from pigs with erysipelas by the Livestock Hygiene Centers from 12 prefectures (Aomori, Niigata, Yamanashi, Ibaraki, Gunma, Nagano, Mie, Gifu, Saitama, Kyoto, Fukuoka and Kumamoto) from 2008 to 2010. Twenty-one isolates originated from acute cases (septicemia), three from sub-acute (urticarial), and 10 from chronic (arthritis and endocarditis) cases (Table 1). All of the cases were identified as E. rhusiopathiae on the basis of cell morphology, characteristic reactions on triple sugar iron agar slants [11] and PCR [12]. Serotyping of the isolates was determined by the gel diffusion precipitin method [13]. Utilization of substrates and enzyme activities of E. rhusiopathiae Met-203 field isolates were confirmed with the rapid ID 32 STREP kit (API System SA, La Balme les Grottes, Montalieu-Vercieu, France). E. rhusiopathiae Fujisawa strain (serotype 1a) was used for the spaA analysis. E. rhusiopathiae Marienfelde strain (serotype 1a) was used for the growth agglutination (GA) test. E. rhusiopathiae were grown in tryptose phosphate broth (TPB) supplemented with 0.1% Tween 80 (pH 7.6) (T-TPB) and agar (TTPA). For the isolation of E. rhusiopathiae from experimentally infected animals, selective media were also used. Selective media or agar was further supplemented with kanamycin (500 mg/ml) and gentamicin (25 mg/ml) [14]. 2.2. Vaccines used Lyophilized live vaccine prepared from acriflavine-fast attenuated E. rhusiopathiae Koganei 65-0.15 (serotype 1a) strain was used for the mouse and swine protection test. For the inactivated
Table 1 Serotypes and origins of the 34 E. rhusiopathiae strains isolated from pigs in Japan during the years 2008e2010. Origin
No. of isolates of serotype
Total
1a
1b
2
10
11
Untypable
Septicemia Urticaria Arthritis Endocarditis
19 0 5 1
0 0 2 0
1 1 2 0
0 1 0 0
0 1 0 0
1 0 0 0
21 3 9 1
Total
25
2
4
1
1
1
34
vaccines, we selected three types of aqueous adjuvant vaccine: a formalin-inactivated whole cell (serotype 2) vaccine with aluminum gel adjuvant, an alkali-extracted antigen (serotype 2) vaccine with a-tocopherol adjuvant, and a recombinant vaccine integrated with surface antigen protein gene of E. rhusiopathiae with aluminum gel adjuvant. All the vaccines are commercially available in Japan. 2.3. Sequence of spaA genes DNA extractions of E. rhusiopathiae isolates were performed as previously described [15]. A 432-bp fragment of the N-terminal half of the spaA gene was PCR amplified and directly sequenced by the modified method of Nagai et al. [16]. Briefly, Primers Erko-1F and Erko-2R were used to amplify the 432-bp fragment. PCR was performed as described previously [17]. PCR products were purified with QIAquick PCR Purification Kit (QIAGEN K.K. Tokyo, Japan). Sequencing reactions of the purified PCR products were performed with primer Erko-1F and primer Erko-2R. The DNA sequences were determined directly by using the BigDye v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., Tokyo, Japan) and an automated DNA sequencer (Applied Biosystems 3130 Genetic Analyzers; Applied Biosystems Inc., Tokyo, Japan). DNA data were analyzed by the GENETYX Network ver.9.0.4. 2.4. Pathogenicity tests in mice Among the spaA variant field isolates, four E. rhusiopathiae Met203 isolates from different areas (Gifu, Gunma, Nagano and Ibaraki Prefectures) were selected and assessed: pathogenicity was determined by their LD50. SPF female ddY mice aged 4e5 weeks were used for the mice experiments. Aliquots (0.1 ml) of serial 10-fold dilutions of T-TPB culture of each isolate were injected subcutaneously into each of five mice [14]. Mortality rates were recorded 14 days after the exposure. The LD50 values were determined by using the method of Karber [18]. 2.5. Mouse protection test One live and three types of inactivated E. rhusiopathiae vaccine were studied for their efficacy against a spaA variant field isolate in the mouse. Mouse protection tests were performed according to the potency test of swine erysipelas vaccines described in the National Assay Standards of Veterinary Biological Products, with the exception of the challenge strain, which was the E. rhusiopathiae Met-203 isolate showing the lowest LD50 instead of the Fujisawa strain. Each of ten ddY mice was vaccinated subcutaneously once or twice according to the standards. Both vaccinated and nonvaccinated mice were challenged 14 days after the last inoculation, with 0.1 ml of the Met-203 field isolate (1.0 102 CFU/mouse). Mouse mortality was monitored for the following 14 days. 2.6. Vaccination and challenge exposure in pigs Three castrated male pigs, (LWD: Landrace, White Yorkshire Duroc), 3e4 months old, were used in our experiments. Two pigs were vaccinated once subcutaneously with 1 ml of the live vaccine; the remaining pig was not vaccinated and acted as a control. Pigs were all challenged intradermally in the flank with 0.1 ml of the Met-203 field isolate (1.4 108 CFU/head) 21 days after the immunization. The clinical responses were observed and the rectal temperature was measured from 5 days before the challenge exposure to 7 days after the challenge exposure. Serum samples were collected from all the pigs at the points of pre- and 14 days after vaccination, and pre- and 14 days after challenge.
M. Uchiyama et al. / Biologicals 42 (2014) 109e113
111
After the challenge exposure, pigs were necropsied on the day of death, or euthanized and necropsied at the end of the experiment. For bacterial isolation, samples were collected from liver, spleen, kidney, heart, lung, internal iliac lymph nodes, tonsils, skin, and joint liquid. Isolation and serotyping of the bacteria were performed using the methods of Takahashi et al. [14], and the sequence of the spaA gene fragment of the isolated bacteria was confirmed.
and Ile-257 classified as Group 3. The isolates in Group 1 with Met-203 and those in Group 3 with Ala-195 displayed characteristic variations of the spaA hypervariable region. Serotype and original clinical signs produced by the different isolates are shown in Table 3. Almost all of the isolates that caused septicemia were classified in Group 1. Eighteen of these were serotype 1a and one was untypable. Group 1 isolates originated from eight prefectures, from both northern and southern areas in Japan. Clinical features caused by Group 2 isolates varied more widely, showing serotypes 1a, 1b, 2, 10 and 11 and producing septicemia, arthritis and urticaria. Group 3 isolates were all serotype 1a, but produced a variety of symptoms including septicemia, arthritis and endocarditis.
2.8. Histology
3.2. Pathogenicity tests in mice
Sampled tissues (heart, lung, liver, spleen, kidney, lymph nodes and tonsils) from all three challenged pigs were fixed in 10% neutral buffered formalin, embedded in paraffin, and sliced in 4 mm sections. Paraffin sections were stained with hematoxylin and eosin. Selected sections were also stained with phosphotungstic acid hematoxylin stain (PTAH) and subjected to further immunohistochemical examination. All immunohistochemical (IHC) examinations were performed using the Envision þ kit (Dako, Carpinteria, CA, U.S.A.) according to the manufacturer’s protocol. The primary antibody was anti-E. rhusiopathiae serotype 1a poly-clonal rabbit serum (1:100 dilution).
The LD50 values of the four E. rhusiopathiae Met-203 field isolates (Group 1) of Gifu, Gunma, Nagano and Ibaraki were 1.45, 1.18, 0.45 and 0.88 CFU/mouse respectively; and the isolate from Nagano, which showed the lowest LD50, was selected for the challenge strain.
All the animal studies were conducted in accordance with the National Veterinary Assay Laboratory Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Acts. 2.7. Necropsy and isolation of E. rhusiopathiae
2.9. Growth agglutination (GA) test For assessing the anti-erysipelas antibody titers, the GA test was performed by a modified method of Sawada et al. [19]. Briefly, sera were treated with 0.2 M 2-mercaptethanol, diluted with TPB and 1 ml of each serum dilution was mixed with 0.025 ml of T-TPB culture of the E. rhusiopathiae Marienfelde strain. The mixture was incubated at 37 C for 18 h. After incubation, the titers were expressed as the reciprocal of the highest dilution of serum that showed signs of agglutination. 3. Results 3.1. Sequence of spaA genes The sequences of the 432-bp hypervariable region of the spaA gene of the 34 field isolates were compared with that of the E. rhusiopathiae Fujisawa strain (Table 2). Based on the sequence analysis of this fragment, the 34 field isolates could be divided into three groups as follows: (i) 19 isolates with Met-203 and isoleucine at position 257 (Ile-257) classified as Group 1, (ii) 12 isolates with Ile-257 classified as Group 2, and (iii) three isolates with Ala-195
Table 2 Substitutions in nucleotide and amino acids in a 432-bp hypervariable region on the spa gene of 34 E. rhusiopathiae field isolates compared with the corresponding sequence of E. rhusiopathiae Fujisawa strain. Strain or group (no. of isolates)
E. rhusiopathiae Fujisawa Group 1 (19) Group 2 (12) Group 3 (3) a
3.3. Mouse protection test E. rhusiopathiae vaccines were all effective in the mouse protection test. When the mice were challenged with approximately 200 LD50 (1.0 102 CFU/mouse) of E. rhusiopathiae Met-203 isolate, survival rates were 100% for all mice that had been immunized with the live vaccine, whole cell inactivated vaccine or recombinant vaccine, and 90% for mice immunized with the alkali-extracted antigen vaccine. Non-vaccinated control mice all died within 5 days of the challenge. The differences of the survival rates between the vaccinated groups and the non-vaccinated group were all significant (P < 0.0001) by the Fisher exact test. 3.4. Vaccination and challenge in pigs The results of challenge in the pigs are shown in Table 4. During the examination, vaccinated pigs did not show any clinical signs except for a red patch at the injection site on the 2nd or 3rd day after vaccination. After challenge with about 1.4 108 CFU/head of an E. rhusiopathiae Met-203 field isolate, the non-vaccinated pig showed depression, anorexia, pyrexia (above 41.5 C) and viscous diarrhea. Urticarial lesions, appearing as purplish red regions (maximum size of erythema: 6.5 cm 5.5 cm) at the injection site, were observed on the 1st day after challenge exposure. The nonvaccinated pig died on the 3rd day after challenge.
Table 3 Serotype and clinical features of E. rhusiopathiae isolates in each spaA group. Group
Serotype
Clinical features (no. of strains)
Group 1 (Met-203)
1a Untypable
Septicemia (18) Septicemia (1)
Group 2
1a 1b 2
Arthritis (4) Arthritis (2) Septicemia (1), Urticaria (1), Arthritis (2) Urticaria (1) Urticaria (1)
Substitutions position of nucleotide (amino acid position)a Nucleotide (aa 195)
Nucleotide (aa 203)
Nucleotide (aa 257)
GAT (Asp)
ATT (Ile)
CTT (Leu)
GAT (Asp) GAT (Asp) GCT (Ala)
ATG (Met) ATT (Ile) ATT (Ile)
ATT (Ile) ATT (Ile) ATT (Ile)
10 11 Group 3
Asp: aspartic acid; Ala: alanine; Ile: isoleucine; Met: methionine; Leu: leucine.
1a
Septicemia (1), Arthritis (1), Endocarditis (1)
E. rhusiopathiae isolates originated from eight prefectures (Aomori, Gunma, Ibaraki, Saitama, Yamanashi, Nagano, Gifu and Fukuoka) for Group 1, two prefectures each for Group 2 (Niigata and Mie) and Group 3 (Kyoto and Kumamoto).
112
M. Uchiyama et al. / Biologicals 42 (2014) 109e113
Table 4 Clinical responses of pigs challenged with E. rhusiopathiae Met-203. Pig no.a
P22 P23 P24
Response after challenge
Growth agglutinating antibody titer
Pyrexia ( C)b
Erythema (cm cm)c
Mortalityc
Before vaccination
14 days after vaccination
Before challenge
14days after challenge
e e 41.5 (2 days after challenge)
e e 6.5 5.5d (2 days after challenge)
e e Dead (3 days after challenge)
&4 &4 &4
128 32
Contents lists available at ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Prevalence of Met-203 type spaA variant in Erysipelothrix rhusiopathiae isolates and the efficacy of swine erysipelas vaccines in Japan Mariko Uchiyama a, Kinya Yamamoto b, Mariko Ochiai a, Tsukasa Yamamoto a, Fumiya Hirano a, Saiki Imamura a, Hidetaka Nagai a, Kouji Ohishi a, Noriyuki Horiuchi c, Mayumi Kijima a, * a
National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, 1-15-1 Tokura, Kokubunji, Tokyo 185-8511, Japan Animal Products Safety Division, Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, 1-2-1 Kasumigaseki, Chiyoda-ku, Tokyo 100-8950, Japan c Department of Basic Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido 080-8555, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 June 2013 Received in revised form 5 November 2013 Accepted 2 December 2013
Since 2009, erysipelas infection among pigs in Japan has been increasing. This study investigated the prevalence, and characteristics of Erysipelothrix rhusiopathiae isolates in Japan from 2008 to 2010 and assessed the efficacy of current commercial erysipelas vaccines. Based on polymorphisms in a 432-bp hypervariable region in the surface protective antigen A (spaA) gene, 34 isolates were classified into three groups: (i) Group 1 with methionine at position 203 (Met-203) and isoleucine at position 257 (Ile257) (18 isolates of serotype 1a and one untypable isolate). (ii) Group 2 with Ile-257 (12 isolates of serotypes 1a, 1b, 2, 10 and 11), and (iii) Group 3 with alanine at position 195 (Ala-195) and Ile-257 (three isolates of serotype 1a). Isolates with Met-203 were highly pathogenic in mice and pigs, causing death in the pig and LD50 values of 0.45e1.45 CFU per mouse. One live and three inactivated commercial E. rhusiopathiae vaccines were evaluated for efficacy against a Met-203 isolate. Almost all mice and pigs that received vaccine survived, while non-vaccinated controls all died within 5 days of the challenge. This indicates that swine erysipelas vaccines might be still effective in protecting animals against the recently prevalent Met-203 isolates in Japan. Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Erysipelothrix rhusiopathiae Vaccine spaA
1. Introduction Erysipelothrix rhusiopathiae is a Gram-positive bacillus that causes erysipelas in a variety of mammals and birds, and dermatological erysipeloid disease in humans [1,2]. Erysipelas is commonly associated with swine, and can be divided into three types by its clinical signs: acute (septicemia), sub-acute (urticaria), and chronic (arthritis, lymphadenitis, and endocarditis). Acute swine erysipelas is characterized by generalized septicemia often resulting in sudden death, and is specified as a notifiable infectious disease in Japan. Pigs infected with erysipelas are prohibited from being slaughtered or are condemned at the slaughterhouse by the Slaughterhouse Act, to prevent human consumption. The pig industry therefore suffers considerable economic loss from this disease. * Corresponding author. Tel.: þ81 42 321 1841; fax: þ81 42 321 1769. E-mail address: [email protected] (M. Kijima).
For the prevention of swine erysipelas, attenuated live vaccine prepared from the acriflavine-fast attenuated Koganei strain 65e 0.15 of E. rhusiopathiae has been used in Japan for many years because of its low-cost and convenience. Appropriate inoculation with the live vaccine produces a strong immunity [3,4]. An inactivated vaccine was approved in 1997 because the live vaccine had a higher incidence of adverse events in pigs kept in a clean environment, such as specific-pathogen-free (SPF) pigs [5,6]. Several kinds of inactivated vaccines have been approved in Japan for swine erysipelas, and their use has been increasing in recent years. The surface protective antigen (Spa) protein of E. rhusiopathiae has been shown to be highly immunogenic and is a promising vaccine candidate for erysipelas [7,8]. The Spa proteins of E. rhusiopathiae can be classified into 3 molecular species, named SpaA (including serotypes 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17 and N), SpaB (serotypes 4, 6, 11, 19 and 21) and SpaC (serotype 18) [8]. A mouse cross-protection study showed that three recombinant Spa proteins (rSpaA, rSpaB and rSpaC) elicited complete protection against
1045-1056/$36.00 Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biologicals.2013.12.002
110
M. Uchiyama et al. / Biologicals 42 (2014) 109e113
challenge with homologous strains but that the level of protection against heterologous strains varied depending on the rSpa protein used for immunization [8]. This indicated that the Spa proteins, especially the N-terminal half of the hyper-variable region, are important for specific immunity. A recent report by To et al. [9] demonstrated that a variant isolate with aspartic acid (Asp) and methionine (Met) at the amino acid positions 195 (Asp-195) and 203 (Met-203), respectively, in the hyper-variable region of the spaA gene might be widespread among pig populations in Japan. The Met-203 isolates, therefore, differ in nucleotide sequence, in the 432-bp hypervariable region of the spaA gene, from the live vaccine strain Koganei 65-0.15 [9]. Unfortunately, according to national statistics, the morbidity rate of swine erysipelas in Japan has been increasing since 2009 [10]. The aim of this study was to characterize recently isolated E. rhusiopathiae strains, assess the prevalence and pathogenicity of the spaA variants, and re-evaluate the validity of swine erysipelas vaccines against a spaA variant field isolate. 2. Materials and methods 2.1. Bacterial strains and growth conditions A total of 34 E. rhusiopathiae isolates were collected for this study. They were isolated from pigs with erysipelas by the Livestock Hygiene Centers from 12 prefectures (Aomori, Niigata, Yamanashi, Ibaraki, Gunma, Nagano, Mie, Gifu, Saitama, Kyoto, Fukuoka and Kumamoto) from 2008 to 2010. Twenty-one isolates originated from acute cases (septicemia), three from sub-acute (urticarial), and 10 from chronic (arthritis and endocarditis) cases (Table 1). All of the cases were identified as E. rhusiopathiae on the basis of cell morphology, characteristic reactions on triple sugar iron agar slants [11] and PCR [12]. Serotyping of the isolates was determined by the gel diffusion precipitin method [13]. Utilization of substrates and enzyme activities of E. rhusiopathiae Met-203 field isolates were confirmed with the rapid ID 32 STREP kit (API System SA, La Balme les Grottes, Montalieu-Vercieu, France). E. rhusiopathiae Fujisawa strain (serotype 1a) was used for the spaA analysis. E. rhusiopathiae Marienfelde strain (serotype 1a) was used for the growth agglutination (GA) test. E. rhusiopathiae were grown in tryptose phosphate broth (TPB) supplemented with 0.1% Tween 80 (pH 7.6) (T-TPB) and agar (TTPA). For the isolation of E. rhusiopathiae from experimentally infected animals, selective media were also used. Selective media or agar was further supplemented with kanamycin (500 mg/ml) and gentamicin (25 mg/ml) [14]. 2.2. Vaccines used Lyophilized live vaccine prepared from acriflavine-fast attenuated E. rhusiopathiae Koganei 65-0.15 (serotype 1a) strain was used for the mouse and swine protection test. For the inactivated
Table 1 Serotypes and origins of the 34 E. rhusiopathiae strains isolated from pigs in Japan during the years 2008e2010. Origin
No. of isolates of serotype
Total
1a
1b
2
10
11
Untypable
Septicemia Urticaria Arthritis Endocarditis
19 0 5 1
0 0 2 0
1 1 2 0
0 1 0 0
0 1 0 0
1 0 0 0
21 3 9 1
Total
25
2
4
1
1
1
34
vaccines, we selected three types of aqueous adjuvant vaccine: a formalin-inactivated whole cell (serotype 2) vaccine with aluminum gel adjuvant, an alkali-extracted antigen (serotype 2) vaccine with a-tocopherol adjuvant, and a recombinant vaccine integrated with surface antigen protein gene of E. rhusiopathiae with aluminum gel adjuvant. All the vaccines are commercially available in Japan. 2.3. Sequence of spaA genes DNA extractions of E. rhusiopathiae isolates were performed as previously described [15]. A 432-bp fragment of the N-terminal half of the spaA gene was PCR amplified and directly sequenced by the modified method of Nagai et al. [16]. Briefly, Primers Erko-1F and Erko-2R were used to amplify the 432-bp fragment. PCR was performed as described previously [17]. PCR products were purified with QIAquick PCR Purification Kit (QIAGEN K.K. Tokyo, Japan). Sequencing reactions of the purified PCR products were performed with primer Erko-1F and primer Erko-2R. The DNA sequences were determined directly by using the BigDye v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., Tokyo, Japan) and an automated DNA sequencer (Applied Biosystems 3130 Genetic Analyzers; Applied Biosystems Inc., Tokyo, Japan). DNA data were analyzed by the GENETYX Network ver.9.0.4. 2.4. Pathogenicity tests in mice Among the spaA variant field isolates, four E. rhusiopathiae Met203 isolates from different areas (Gifu, Gunma, Nagano and Ibaraki Prefectures) were selected and assessed: pathogenicity was determined by their LD50. SPF female ddY mice aged 4e5 weeks were used for the mice experiments. Aliquots (0.1 ml) of serial 10-fold dilutions of T-TPB culture of each isolate were injected subcutaneously into each of five mice [14]. Mortality rates were recorded 14 days after the exposure. The LD50 values were determined by using the method of Karber [18]. 2.5. Mouse protection test One live and three types of inactivated E. rhusiopathiae vaccine were studied for their efficacy against a spaA variant field isolate in the mouse. Mouse protection tests were performed according to the potency test of swine erysipelas vaccines described in the National Assay Standards of Veterinary Biological Products, with the exception of the challenge strain, which was the E. rhusiopathiae Met-203 isolate showing the lowest LD50 instead of the Fujisawa strain. Each of ten ddY mice was vaccinated subcutaneously once or twice according to the standards. Both vaccinated and nonvaccinated mice were challenged 14 days after the last inoculation, with 0.1 ml of the Met-203 field isolate (1.0 102 CFU/mouse). Mouse mortality was monitored for the following 14 days. 2.6. Vaccination and challenge exposure in pigs Three castrated male pigs, (LWD: Landrace, White Yorkshire Duroc), 3e4 months old, were used in our experiments. Two pigs were vaccinated once subcutaneously with 1 ml of the live vaccine; the remaining pig was not vaccinated and acted as a control. Pigs were all challenged intradermally in the flank with 0.1 ml of the Met-203 field isolate (1.4 108 CFU/head) 21 days after the immunization. The clinical responses were observed and the rectal temperature was measured from 5 days before the challenge exposure to 7 days after the challenge exposure. Serum samples were collected from all the pigs at the points of pre- and 14 days after vaccination, and pre- and 14 days after challenge.
M. Uchiyama et al. / Biologicals 42 (2014) 109e113
111
After the challenge exposure, pigs were necropsied on the day of death, or euthanized and necropsied at the end of the experiment. For bacterial isolation, samples were collected from liver, spleen, kidney, heart, lung, internal iliac lymph nodes, tonsils, skin, and joint liquid. Isolation and serotyping of the bacteria were performed using the methods of Takahashi et al. [14], and the sequence of the spaA gene fragment of the isolated bacteria was confirmed.
and Ile-257 classified as Group 3. The isolates in Group 1 with Met-203 and those in Group 3 with Ala-195 displayed characteristic variations of the spaA hypervariable region. Serotype and original clinical signs produced by the different isolates are shown in Table 3. Almost all of the isolates that caused septicemia were classified in Group 1. Eighteen of these were serotype 1a and one was untypable. Group 1 isolates originated from eight prefectures, from both northern and southern areas in Japan. Clinical features caused by Group 2 isolates varied more widely, showing serotypes 1a, 1b, 2, 10 and 11 and producing septicemia, arthritis and urticaria. Group 3 isolates were all serotype 1a, but produced a variety of symptoms including septicemia, arthritis and endocarditis.
2.8. Histology
3.2. Pathogenicity tests in mice
Sampled tissues (heart, lung, liver, spleen, kidney, lymph nodes and tonsils) from all three challenged pigs were fixed in 10% neutral buffered formalin, embedded in paraffin, and sliced in 4 mm sections. Paraffin sections were stained with hematoxylin and eosin. Selected sections were also stained with phosphotungstic acid hematoxylin stain (PTAH) and subjected to further immunohistochemical examination. All immunohistochemical (IHC) examinations were performed using the Envision þ kit (Dako, Carpinteria, CA, U.S.A.) according to the manufacturer’s protocol. The primary antibody was anti-E. rhusiopathiae serotype 1a poly-clonal rabbit serum (1:100 dilution).
The LD50 values of the four E. rhusiopathiae Met-203 field isolates (Group 1) of Gifu, Gunma, Nagano and Ibaraki were 1.45, 1.18, 0.45 and 0.88 CFU/mouse respectively; and the isolate from Nagano, which showed the lowest LD50, was selected for the challenge strain.
All the animal studies were conducted in accordance with the National Veterinary Assay Laboratory Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Acts. 2.7. Necropsy and isolation of E. rhusiopathiae
2.9. Growth agglutination (GA) test For assessing the anti-erysipelas antibody titers, the GA test was performed by a modified method of Sawada et al. [19]. Briefly, sera were treated with 0.2 M 2-mercaptethanol, diluted with TPB and 1 ml of each serum dilution was mixed with 0.025 ml of T-TPB culture of the E. rhusiopathiae Marienfelde strain. The mixture was incubated at 37 C for 18 h. After incubation, the titers were expressed as the reciprocal of the highest dilution of serum that showed signs of agglutination. 3. Results 3.1. Sequence of spaA genes The sequences of the 432-bp hypervariable region of the spaA gene of the 34 field isolates were compared with that of the E. rhusiopathiae Fujisawa strain (Table 2). Based on the sequence analysis of this fragment, the 34 field isolates could be divided into three groups as follows: (i) 19 isolates with Met-203 and isoleucine at position 257 (Ile-257) classified as Group 1, (ii) 12 isolates with Ile-257 classified as Group 2, and (iii) three isolates with Ala-195
Table 2 Substitutions in nucleotide and amino acids in a 432-bp hypervariable region on the spa gene of 34 E. rhusiopathiae field isolates compared with the corresponding sequence of E. rhusiopathiae Fujisawa strain. Strain or group (no. of isolates)
E. rhusiopathiae Fujisawa Group 1 (19) Group 2 (12) Group 3 (3) a
3.3. Mouse protection test E. rhusiopathiae vaccines were all effective in the mouse protection test. When the mice were challenged with approximately 200 LD50 (1.0 102 CFU/mouse) of E. rhusiopathiae Met-203 isolate, survival rates were 100% for all mice that had been immunized with the live vaccine, whole cell inactivated vaccine or recombinant vaccine, and 90% for mice immunized with the alkali-extracted antigen vaccine. Non-vaccinated control mice all died within 5 days of the challenge. The differences of the survival rates between the vaccinated groups and the non-vaccinated group were all significant (P < 0.0001) by the Fisher exact test. 3.4. Vaccination and challenge in pigs The results of challenge in the pigs are shown in Table 4. During the examination, vaccinated pigs did not show any clinical signs except for a red patch at the injection site on the 2nd or 3rd day after vaccination. After challenge with about 1.4 108 CFU/head of an E. rhusiopathiae Met-203 field isolate, the non-vaccinated pig showed depression, anorexia, pyrexia (above 41.5 C) and viscous diarrhea. Urticarial lesions, appearing as purplish red regions (maximum size of erythema: 6.5 cm 5.5 cm) at the injection site, were observed on the 1st day after challenge exposure. The nonvaccinated pig died on the 3rd day after challenge.
Table 3 Serotype and clinical features of E. rhusiopathiae isolates in each spaA group. Group
Serotype
Clinical features (no. of strains)
Group 1 (Met-203)
1a Untypable
Septicemia (18) Septicemia (1)
Group 2
1a 1b 2
Arthritis (4) Arthritis (2) Septicemia (1), Urticaria (1), Arthritis (2) Urticaria (1) Urticaria (1)
Substitutions position of nucleotide (amino acid position)a Nucleotide (aa 195)
Nucleotide (aa 203)
Nucleotide (aa 257)
GAT (Asp)
ATT (Ile)
CTT (Leu)
GAT (Asp) GAT (Asp) GCT (Ala)
ATG (Met) ATT (Ile) ATT (Ile)
ATT (Ile) ATT (Ile) ATT (Ile)
10 11 Group 3
Asp: aspartic acid; Ala: alanine; Ile: isoleucine; Met: methionine; Leu: leucine.
1a
Septicemia (1), Arthritis (1), Endocarditis (1)
E. rhusiopathiae isolates originated from eight prefectures (Aomori, Gunma, Ibaraki, Saitama, Yamanashi, Nagano, Gifu and Fukuoka) for Group 1, two prefectures each for Group 2 (Niigata and Mie) and Group 3 (Kyoto and Kumamoto).
112
M. Uchiyama et al. / Biologicals 42 (2014) 109e113
Table 4 Clinical responses of pigs challenged with E. rhusiopathiae Met-203. Pig no.a
P22 P23 P24
Response after challenge
Growth agglutinating antibody titer
Pyrexia ( C)b
Erythema (cm cm)c
Mortalityc
Before vaccination
14 days after vaccination
Before challenge
14days after challenge
e e 41.5 (2 days after challenge)
e e 6.5 5.5d (2 days after challenge)
e e Dead (3 days after challenge)
&4 &4 &4
128 32
