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Serological diversity and virulence determination of Dichelobacter nodosus from footrot in India
Molecular and Cellular Probes 23 (2009) 112–114
Contents lists available at ScienceDirect
Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr
Serological diversity and virulence determination of Dichelobacter nodosus from footrot in India I. Hussain, S.A. Wani*, S.D. Qureshi, S. Farooq Bacteriology Laboratory, Division of Veterinary Microbiology and Immunology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shuhama (Alusteng), Srinagar 190006, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 July 2008 Accepted 7 January 2009 Available online 21 January 2009
One hundred and twenty-eight swab samples from footrot lesions of naturally infected sheep were examined for presence of Dichelobacter nodosus (D. nodosus). The detection of D. nodosus was carried out by polymerase chain reaction (PCR), directly from swabs or after isolation, using 16S rDNA specific primers. The isolation of the bacterium was carried out anaerobically on trypticase-arginine-serine (TAS) agar containing 4% hoof powder. Serogrouping of the D. nodosus was accomplished with multiplex PCR using nine (A–I) serogroup specific primers. The virulent and benign status of the isolates was ascertained by detection of virulence specific integrase A (intA) gene. Out of total 83 D. nodosus isolates, 62 (74.69%) belonged to serogroup B, 18 (21.68%) to serogroup E and three (3.62%) to serogroup I. Serogroup I was detected and isolated for the first time in India. All the positive samples revealed infection by single serogroup of D. nodosus except one which showed mixed infection of serogroups B and E. Sixty (72.28%) isolates possessed intA gene and thus were considered as virulent strains. Serogroupwise intA gene was found in 43 (69.35%) isolates of serogroup B, 14 (77.78%) of E and in all the three (100%) of I. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Footrot India PCR Serogroup intA
1. Introduction Footrot is an economically important disease of sheep and goats that is caused by the synergistic action of several bacterial species, of which Dichelobacter nodosus (D. nodosus) is the essential causative agent [1]. D. nodosus is a strictly anaerobic bacterium comprised of a number of serogroups based on its fimbrial antigen [2]. The current Australian classification system classifies D. nodosus into at least 10 serogroups (A–I and M) [3,4]. Previously, identification of serogroups of D. nodosus in a footrot lesion required isolation of the organism, purification by subculture followed by antigenic analysis with serological methods, which could take 3–4 weeks. Currently, identification of the D. nodosus is being carried out by polymerase chain reaction (PCR) using 16S rDNA specific primers [5] while its serogouping is being carried out by multiplex PCR (m-PCR) [6] using serogroup-specific primers without the need to culture. D. nodosus strains can be categorized as virulent, intermediate and benign, based on the corresponding forms of the disease [1]. Earlier strain differentiation of D. nodosus was carried out by gelatin gel test [7] based on their production of thermostable proteases. However, it was observed that some strains of D. nodosus
* Corresponding author. Tel./fax: þ91 194 2262211. E-mail address: [email protected] (S.A. Wani). 0890-8508/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2009.01.003
which secrete thermostable proteases are incapable of causing virulent footrot [8]. Subsequently, DNA probes like vap and vrl [9,10] and specific primers for PCR [11] were developed for differentiation of virulent and benign strains of D. nodosus. Recently, Cheetham et al [8] described a PCR assay for detection of the integrase (intA) gene, a virulent specific genetic element, that was found to be superior to earlier methods. In our previous studies [12,13], we reported detection and/or isolation of serogroup B and E of D. nodosus from footrot lesions of sheep and goats in India. The objective of the present investigation was to expand our earlier findings for better understanding of the epidemiology and serological diversity of D. nodosus as well as to ascertain the virulent or benign status of the strains on the basis of presence or absence of intA gene. 2. Materials and methods 2.1. Collection of clinical samples Exudates of footrot lesions with a lesion score of 2 (interdigital dermatitis)–4 (underrunning of the hard horn of the hoof) [14] were collected from 128 naturally infected sheep of Govt. Sheep Breeding Farm, Goabal and of private owners from the Ganderbal, Kangan and Sonamarg area of the Kashmir valley during December 2006–March 2008. Samples were collected on sterile cotton swabs
I. Hussain et al. / Molecular and Cellular Probes 23 (2009) 112–114 Table 1 Correlation between direct and culture PCR for detection and serogrouping of D. nodosus and serogroupwise distribution of intA gene. Sample No. of Serogroups samples tested/DN B E positive
Serogroupwise intA positive I
Untyped B
Swabs 128/67a
50 (74.63) 13 (19.40) 3 (4.47) 2
Culture 128/82a
62 (75.61) 18 (21.95) 3 (3.66) 0
Not done 43 (69.35)
E
I
Not done 14 (77.378)
Not done 3 (100)
DN ¼ D. nodosus. a One sample revealed mixed infection with B and E serogroups. Figures in parentheses indicate percentage.
in duplicate, one for inoculation into media and another for DNA extraction. 2.2. Extraction of bacterial DNA Suspensions of the material present on the swabs were prepared in 1.5 ml microcentrifuge tubes in 100 ml of sterile phosphate buffered saline (PBS) by gentle vortexing. After removing the swabs, the samples were boiled for 5 min, cooled on ice for 10 min and centrifuged at 10,000 g for 1 min. Similarly, from the culture plates suspected colonies with characteristic morphology were directly suspended into 100 ml of sterile PBS and processed for the extraction of DNA as before. Two microlitres of the supernatant was used as the template for each PCR reaction. 2.3. Detection of 16S rRNA gene of D. nodosus by PCR For the detection of 16S rRNA gene of D. nodosus PCR conditions and primers were essentially the same as described earlier [5]. Positive control DNA kindly supplied by Dr. O. P. Dhungyel, Faculty of Veterinary Medicine, University of Sydney, Cambden, NSW 2570, Australia, was included in the PCR. Distilled water served as negative control. 2.4. Isolation and serogrouping of D. nodosus For isolation of D. nodosus, all the swab samples were streaked at the place of collection on tripticase-arginine-serine (TAS) agar with 4% hoof powder [15]. The hoof powder was prepared from dried hooves collected from healthy slaughtered sheep. After inoculation, the plates were placed immediately in an anaerobic jar with Anaerogaspack (HiMedia, Mumbai, India) and carried to the laboratory. The jar was placed in incubator at 37 C within 2–3 h. After 5 days of incubation, suspected colonies [15] were subcultured on the
113
same medium until they were free from contaminating bacteria. They were then subcultured on TAS medium containing 2% hoof powder until pure colonies of D. nodosus were obtained. Plates, which did not show any characteristics growth after initial 5 days of incubation, were incubated for another 5 days before being discarded. Confirmation of the isolates as D. nodosus was done by demonstration of the typical cellular morphology in Gram stained smears and detection of species-specific 16S rRNA gene by PCR as described above except the hoof medium was used as a negative control. For serogrouping multiplex PCR was carried out by using nine (A–I) serogroup specific primers as described earlier [6]. Serogroup specific DNA kindly supplied by Dr. O. P. Dhungyel, was used as positive control in PCR. 2.5. Strain differentiation of D. nodosus into virulent and benign To ascertain the virulent or benign status, all the D. nodosus isolates were screened for presence of intA gene. Detection of intA gene was carried out as per the method described earlier [8] with minor modifications. The concentration of the primer pair (50 ACA TCA TGC GAC TCA CTG AC30 and 50 TCT CTG GTC GGT CGT ACA AT 30 ) was 0.25 mM while concentration of MgCl2 was 2.0 mM. The amplification was done with 31 cycles, each cycle consisting of 1.5 min at 94 C, 1 min at 60 C and 2 min at 72 C. This was followed by a final extension of 5 min at 72 C. 2.6. Analysis of PCR products The PCR products were analysed in 1–2% agarose gels, stained with ethidium bromide, visualized under ultraviolet illumination and photographed. 3. Results Out of 128 swab samples, 67 (Table 1) yielded an amplicon of 783 bp conforming to the expected size of 16S rDNA of D. nodosus in the PCR reactions. However, out of 128 swab samples, 82 revealed D. nodosus by anaerobic culture. Out of 82 samples, one yielded mixed infection of two serogroups. Sixteen samples negative by direct PCR revealed presence of D. nodosus after culture. However, one sample positive by direct PCR did not yield D. nodosus on culture. The m-PCR assay for serogrouping was successfully applied to all the swab specimens except two. However, all the 83 isolates were serogrouped by m-PCR (Table 1). Out of 67 swab samples positive for D. nodosus, 49 yielded a single band of 283 bp characteristic of serogroup B, 12 samples produced a serogroup E specific band of 363 bp and three samples revealed a single band of 189 bp which is characteristics of serogroup I. The remaining one sample
Fig. 1. (A) Serogrouping of D. nodosus isolates by multiplex PCR. Lane 1, 2 and 3: samples positive for serogroup I, E and B, respectively. Lane M: 100 bp DNA marker, Lane 4 and 5: positive and negative control, respectively. (B) Detection of intA gene of D. nodosus by PCR. Lane 1: 100 bp DNA marker, Lane 1: sample positive for intA gene, Lane 2 and 3: positive and negative control, respectively.
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yielded both serogroup B and E specific bands. Similarly 62 isolates were serogrouped as B while 18 were serogrouped as E and three belonged to serogroup I (Table 1, Fig. 1A). Only one sample yielded two isolates of serogroups B and E. Serogroups of the isolates were same as detected in their respective swab samples. Serogroup specificity of each isolates was again confirmed by simplex PCR using single pair of primers for the respective serogroups. Out of total 83 isolates, 60 were found to carry the intA gene as they yielded an amplicon of 530 bp (Fig. 1B). Serogroupwise distribution revealed 43 isolates of serogroup B, 14 isolates of serogroup E and three isolates of serogroup I positive for intA gene (Table 1). 4. Discussion and conclusions Footrot has become enzootic in the sheep population in the state of Jammu and Kashmir, for the last 16 years or more [13]. The severity and widespread nature of the disease poses great challenge to sheep rearing in the state and demands the immediate development of control measures including a vaccine. To date, it is a common practice to prepare the commercial footrot vaccine containing representative serogroups available locally. This serogroup-specific vaccination was widely used for successful control and eradication of virulent footrot in different countries including Nepal and Bhutan [16,17]. Thus for the development of an efficacious vaccine against virulent footrot, it is essential to know the serological diversity of the causative agent as well as virulent status of strains. In the present investigation, we have successfully isolated D. nodosus belonging to three different serogroups from 64.06% footrot samples. Although we collected samples from virulent footrot we failed to isolate D. nodosus from 35.94% samples. The possible explanation is that the samples might be having less number of bacteria or the lesions were in the process of healing. In the present study the culture method in conjunction with PCR was found to be more sensitive than by direct PCR in detection of D. nodosus. This corroborated with the findings of Dhungyel et al. [6]. The possible explanation may be due to the crude method of DNA extraction followed by us that may contain PCR inhibitory substances or very less number of D. nodosus cells in the swab [18]. However, detection of D. nodosus by direct PCR is a very rapid test as compared to culture method. Out of 83 isolates, serogroup B was found to be most predominant (74.69%) as compared to serogroup E (21.68%) and thus confirms our earlier findings [12,13]. Similarly, serogroup B was encountered as the predominant serogroup in our neighbouring country Bhutan [17]. However, serogroup E was recorded as the most frequent among Nepalese isolates [19]; it is relatively uncommon in Australia [20], UK [21] and USA [22]. In addition, we also isolated serogroup I for the first time here. However, its prevalence was found to be least as compared to serogroups B and E. This is in agreement with the findings of Moore et al [2] who also reported the presence of serogroup I of D. nodosus in sheep with a similar prevalent rate (3.5%) in England and Wales. Mixed infection with two different serogroups detected in the present study corroborates with the findings of earlier investigators [2]. The detection of serogroup B in majority of the samples and serogroup E in good number of samples suggests that strains of serogroups B and E could be an appropriate candidate for the development of an effective vaccine. However, further studies in this direction covering more number of flocks and geographical area will precisely determine the formulation of an effective vaccine against the disease in this region. In the present study, 72.28% isolates carried the intA gene thus were considered as virulent strains and this explains the virulent nature and severity of lesions of footrot in the affected animals. We failed to isolate intA positive D. nodosus from 27.72% of the samples.
This could be due to that the sheep with virulent footrot may carry benign strains [8]. In conclusion virulent strains of D. nodosus belonging to at least three different serogroups with predominant prevalence of serogroups B and E are causing virulent footrot in sheep population in Kashmir (India). Our findings also suggest the formulation of a bivalent vaccine from serogroups B and E against the menace in the state. Acknowledgements The authors are highly thankful to Dr. O.P. Dhungyel, Faculty of Veterinary Medicine, University of Sydney, Cambden, NSW 2570, Australia, for the supply of serogroup specific positive control DNA. References [1] Stewart D. Footrot in sheep. In: Egerton JR, Yong WK, Riffkin GG, editors. Footrot and foot abscesses of ruminants. Boca Raton, Florida: CRC Press; 1989. p. 5–45. [2] Moore LJ, Wassink GJ, Green LE, Grogono-Thomas R. The detection and characterization of Dichelobacter nodosus from cases of ovine footrot in England and Wales. Vet Microbiol 2005;108:57–67. [3] Claxton PD. Serogrouping of Bacteroides nodosus isolates. In: Stewart DJ, Peterson JE, McKern NM, Emery DL, editors. Footrot in ruminants. Proceedings of a workshop, Melbourne, 1985. Glebe, Sydney, N.S.W, Australia: CSIRO, Division of Animal Health and Australian Wool Corporation; 1986. p. 131–134. [4] Ghimire SC, Egerton JR, Dhungyel OP, Joshi HD. Identification and characterization of serogroup M among Nepalese isolates of Dichelobacter nodosus, the transmitting agent of footrot in small ruminants. Vet Microbiol 1998;62: 217–233. [5] La Fontaine SL, Egerton JR, Rood JI. Detection of Dichelobacter nodosus using species-specific oligonucleotides as PCR primers. Vet Microbiol 1993;35: 101–117. [6] Dhungyel OP, Whittington RJ, Egerton JR. Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination. Mol Cell Probes 2002;16:285–296. [7] Palmer MA. A gelatin test to detect activity and stability of proteases produced by Dichelobacter (Bacteroides) nodosus. Vet Microbiol 1993;36:113–122. [8] Cheetham BF, Tanjung LR, Sutherland M, Druitt J, Green G, McFarlane J, et al. Improved diagnosis of virulent ovine footrot using the intA gene. Vet Microbiol 2006;116:166–174. [9] Katz ME, Howarth PM, Young WK, Rifkin GG, Depiazi LJ, Rood JI. Identification of three gene regions associated with virulence in Dichelobacter nodosus, the causative agent of ovine footrot. J Gen Microbiol 1991;137:2117–2124. [10] Rood JI, Howarth PA, Haring V, Yong WK, Liu D, Palmer MA, et al. Comparison of gene probe and conventional methods for the diagnosis of ovine footrot. Vet Microbiol 1996;52:127–142. [11] Liu D, Webber J. A polymerase chain reaction assay for improved determination of virulence of Dichelobacter nodosus, the specific causative pathogen for ovine footrot. Vet Microbiol 1995;43:197–207. [12] Wani SA, Samanta I, Buchh AS, Bhat MA. Molecular detection and characterization of Dichelobacter nodosus in ovine footrot in India. Mol Cell Probes 2004;18:289–291. [13] Wani SA, Samanta I, Shah SS. Isolation and characterization of Dichelobacter nodosus from ovine and caprine footrot in Kashmir, India. Res Vet Sci 2007;83:141–144. [14] Egerton JR, Roberts DS. Vaccination against ovine footrot. J Comp Pathol 1971;81:179–185. [15] Thorley CM. A simplified method for the isolation of Bacteroides nodosus from ovine footrot and studies on its colony morphology and serology. J Appl Bacteriol 1976;40:301–309. [16] Egerton JR, Ghimire SC, Dhungyel OP, Shrestha HK, Joshi HD, Joshi BR, et al. Eradication of virulent footrot from sheep and goats in an endemic area of Nepal and an evaluation of specific vaccination. Vet Rec 2002;151:290–295. [17] Gurung RB, Dhungyel OP, Tshering P, Egerton JR. The use of an autogenous Dichelobacter nodosus vaccine to eliminate clinical signs of virulent footrot in a sheep flock in Bhutan. Vet J 2006;172:356–363. [18] Cagatay IT, Hickford JGH. Update on ovine footrot in New Zealand: isolation, identification and characterization of Dichelobacter nodosus strains. Vet Microbiol 2005;111:171–180. [19] Ghimire SC, Egerton JR, Dhungyel OP. Characterization of Dichelobacter nodosus isolated from footrot in sheep and goat in Nepal. Small Rumin Res 1996;23:59–67. [20] Claxton PD, Ribeiro LA, Egerton JR. Classification of Bacteroides nodosus by agglutination test. Aust Vet J 1983;70:123–126. [21] Hindmarsh F, Fraser J. Serogrouping of Bacteroides nodosus isolated from ovine footrot in Britain. Vet Rec 1985;116:187–188. [22] Gradin JL, Sonn AE, Petrovska L. Serogrouping of Bacteroides nodosus isolates from 62 sources in the United States. Am J Vet Res 1993;54:1069–1073.
Contents lists available at ScienceDirect
Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr
Serological diversity and virulence determination of Dichelobacter nodosus from footrot in India I. Hussain, S.A. Wani*, S.D. Qureshi, S. Farooq Bacteriology Laboratory, Division of Veterinary Microbiology and Immunology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shuhama (Alusteng), Srinagar 190006, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 July 2008 Accepted 7 January 2009 Available online 21 January 2009
One hundred and twenty-eight swab samples from footrot lesions of naturally infected sheep were examined for presence of Dichelobacter nodosus (D. nodosus). The detection of D. nodosus was carried out by polymerase chain reaction (PCR), directly from swabs or after isolation, using 16S rDNA specific primers. The isolation of the bacterium was carried out anaerobically on trypticase-arginine-serine (TAS) agar containing 4% hoof powder. Serogrouping of the D. nodosus was accomplished with multiplex PCR using nine (A–I) serogroup specific primers. The virulent and benign status of the isolates was ascertained by detection of virulence specific integrase A (intA) gene. Out of total 83 D. nodosus isolates, 62 (74.69%) belonged to serogroup B, 18 (21.68%) to serogroup E and three (3.62%) to serogroup I. Serogroup I was detected and isolated for the first time in India. All the positive samples revealed infection by single serogroup of D. nodosus except one which showed mixed infection of serogroups B and E. Sixty (72.28%) isolates possessed intA gene and thus were considered as virulent strains. Serogroupwise intA gene was found in 43 (69.35%) isolates of serogroup B, 14 (77.78%) of E and in all the three (100%) of I. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Footrot India PCR Serogroup intA
1. Introduction Footrot is an economically important disease of sheep and goats that is caused by the synergistic action of several bacterial species, of which Dichelobacter nodosus (D. nodosus) is the essential causative agent [1]. D. nodosus is a strictly anaerobic bacterium comprised of a number of serogroups based on its fimbrial antigen [2]. The current Australian classification system classifies D. nodosus into at least 10 serogroups (A–I and M) [3,4]. Previously, identification of serogroups of D. nodosus in a footrot lesion required isolation of the organism, purification by subculture followed by antigenic analysis with serological methods, which could take 3–4 weeks. Currently, identification of the D. nodosus is being carried out by polymerase chain reaction (PCR) using 16S rDNA specific primers [5] while its serogouping is being carried out by multiplex PCR (m-PCR) [6] using serogroup-specific primers without the need to culture. D. nodosus strains can be categorized as virulent, intermediate and benign, based on the corresponding forms of the disease [1]. Earlier strain differentiation of D. nodosus was carried out by gelatin gel test [7] based on their production of thermostable proteases. However, it was observed that some strains of D. nodosus
* Corresponding author. Tel./fax: þ91 194 2262211. E-mail address: [email protected] (S.A. Wani). 0890-8508/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2009.01.003
which secrete thermostable proteases are incapable of causing virulent footrot [8]. Subsequently, DNA probes like vap and vrl [9,10] and specific primers for PCR [11] were developed for differentiation of virulent and benign strains of D. nodosus. Recently, Cheetham et al [8] described a PCR assay for detection of the integrase (intA) gene, a virulent specific genetic element, that was found to be superior to earlier methods. In our previous studies [12,13], we reported detection and/or isolation of serogroup B and E of D. nodosus from footrot lesions of sheep and goats in India. The objective of the present investigation was to expand our earlier findings for better understanding of the epidemiology and serological diversity of D. nodosus as well as to ascertain the virulent or benign status of the strains on the basis of presence or absence of intA gene. 2. Materials and methods 2.1. Collection of clinical samples Exudates of footrot lesions with a lesion score of 2 (interdigital dermatitis)–4 (underrunning of the hard horn of the hoof) [14] were collected from 128 naturally infected sheep of Govt. Sheep Breeding Farm, Goabal and of private owners from the Ganderbal, Kangan and Sonamarg area of the Kashmir valley during December 2006–March 2008. Samples were collected on sterile cotton swabs
I. Hussain et al. / Molecular and Cellular Probes 23 (2009) 112–114 Table 1 Correlation between direct and culture PCR for detection and serogrouping of D. nodosus and serogroupwise distribution of intA gene. Sample No. of Serogroups samples tested/DN B E positive
Serogroupwise intA positive I
Untyped B
Swabs 128/67a
50 (74.63) 13 (19.40) 3 (4.47) 2
Culture 128/82a
62 (75.61) 18 (21.95) 3 (3.66) 0
Not done 43 (69.35)
E
I
Not done 14 (77.378)
Not done 3 (100)
DN ¼ D. nodosus. a One sample revealed mixed infection with B and E serogroups. Figures in parentheses indicate percentage.
in duplicate, one for inoculation into media and another for DNA extraction. 2.2. Extraction of bacterial DNA Suspensions of the material present on the swabs were prepared in 1.5 ml microcentrifuge tubes in 100 ml of sterile phosphate buffered saline (PBS) by gentle vortexing. After removing the swabs, the samples were boiled for 5 min, cooled on ice for 10 min and centrifuged at 10,000 g for 1 min. Similarly, from the culture plates suspected colonies with characteristic morphology were directly suspended into 100 ml of sterile PBS and processed for the extraction of DNA as before. Two microlitres of the supernatant was used as the template for each PCR reaction. 2.3. Detection of 16S rRNA gene of D. nodosus by PCR For the detection of 16S rRNA gene of D. nodosus PCR conditions and primers were essentially the same as described earlier [5]. Positive control DNA kindly supplied by Dr. O. P. Dhungyel, Faculty of Veterinary Medicine, University of Sydney, Cambden, NSW 2570, Australia, was included in the PCR. Distilled water served as negative control. 2.4. Isolation and serogrouping of D. nodosus For isolation of D. nodosus, all the swab samples were streaked at the place of collection on tripticase-arginine-serine (TAS) agar with 4% hoof powder [15]. The hoof powder was prepared from dried hooves collected from healthy slaughtered sheep. After inoculation, the plates were placed immediately in an anaerobic jar with Anaerogaspack (HiMedia, Mumbai, India) and carried to the laboratory. The jar was placed in incubator at 37 C within 2–3 h. After 5 days of incubation, suspected colonies [15] were subcultured on the
113
same medium until they were free from contaminating bacteria. They were then subcultured on TAS medium containing 2% hoof powder until pure colonies of D. nodosus were obtained. Plates, which did not show any characteristics growth after initial 5 days of incubation, were incubated for another 5 days before being discarded. Confirmation of the isolates as D. nodosus was done by demonstration of the typical cellular morphology in Gram stained smears and detection of species-specific 16S rRNA gene by PCR as described above except the hoof medium was used as a negative control. For serogrouping multiplex PCR was carried out by using nine (A–I) serogroup specific primers as described earlier [6]. Serogroup specific DNA kindly supplied by Dr. O. P. Dhungyel, was used as positive control in PCR. 2.5. Strain differentiation of D. nodosus into virulent and benign To ascertain the virulent or benign status, all the D. nodosus isolates were screened for presence of intA gene. Detection of intA gene was carried out as per the method described earlier [8] with minor modifications. The concentration of the primer pair (50 ACA TCA TGC GAC TCA CTG AC30 and 50 TCT CTG GTC GGT CGT ACA AT 30 ) was 0.25 mM while concentration of MgCl2 was 2.0 mM. The amplification was done with 31 cycles, each cycle consisting of 1.5 min at 94 C, 1 min at 60 C and 2 min at 72 C. This was followed by a final extension of 5 min at 72 C. 2.6. Analysis of PCR products The PCR products were analysed in 1–2% agarose gels, stained with ethidium bromide, visualized under ultraviolet illumination and photographed. 3. Results Out of 128 swab samples, 67 (Table 1) yielded an amplicon of 783 bp conforming to the expected size of 16S rDNA of D. nodosus in the PCR reactions. However, out of 128 swab samples, 82 revealed D. nodosus by anaerobic culture. Out of 82 samples, one yielded mixed infection of two serogroups. Sixteen samples negative by direct PCR revealed presence of D. nodosus after culture. However, one sample positive by direct PCR did not yield D. nodosus on culture. The m-PCR assay for serogrouping was successfully applied to all the swab specimens except two. However, all the 83 isolates were serogrouped by m-PCR (Table 1). Out of 67 swab samples positive for D. nodosus, 49 yielded a single band of 283 bp characteristic of serogroup B, 12 samples produced a serogroup E specific band of 363 bp and three samples revealed a single band of 189 bp which is characteristics of serogroup I. The remaining one sample
Fig. 1. (A) Serogrouping of D. nodosus isolates by multiplex PCR. Lane 1, 2 and 3: samples positive for serogroup I, E and B, respectively. Lane M: 100 bp DNA marker, Lane 4 and 5: positive and negative control, respectively. (B) Detection of intA gene of D. nodosus by PCR. Lane 1: 100 bp DNA marker, Lane 1: sample positive for intA gene, Lane 2 and 3: positive and negative control, respectively.
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yielded both serogroup B and E specific bands. Similarly 62 isolates were serogrouped as B while 18 were serogrouped as E and three belonged to serogroup I (Table 1, Fig. 1A). Only one sample yielded two isolates of serogroups B and E. Serogroups of the isolates were same as detected in their respective swab samples. Serogroup specificity of each isolates was again confirmed by simplex PCR using single pair of primers for the respective serogroups. Out of total 83 isolates, 60 were found to carry the intA gene as they yielded an amplicon of 530 bp (Fig. 1B). Serogroupwise distribution revealed 43 isolates of serogroup B, 14 isolates of serogroup E and three isolates of serogroup I positive for intA gene (Table 1). 4. Discussion and conclusions Footrot has become enzootic in the sheep population in the state of Jammu and Kashmir, for the last 16 years or more [13]. The severity and widespread nature of the disease poses great challenge to sheep rearing in the state and demands the immediate development of control measures including a vaccine. To date, it is a common practice to prepare the commercial footrot vaccine containing representative serogroups available locally. This serogroup-specific vaccination was widely used for successful control and eradication of virulent footrot in different countries including Nepal and Bhutan [16,17]. Thus for the development of an efficacious vaccine against virulent footrot, it is essential to know the serological diversity of the causative agent as well as virulent status of strains. In the present investigation, we have successfully isolated D. nodosus belonging to three different serogroups from 64.06% footrot samples. Although we collected samples from virulent footrot we failed to isolate D. nodosus from 35.94% samples. The possible explanation is that the samples might be having less number of bacteria or the lesions were in the process of healing. In the present study the culture method in conjunction with PCR was found to be more sensitive than by direct PCR in detection of D. nodosus. This corroborated with the findings of Dhungyel et al. [6]. The possible explanation may be due to the crude method of DNA extraction followed by us that may contain PCR inhibitory substances or very less number of D. nodosus cells in the swab [18]. However, detection of D. nodosus by direct PCR is a very rapid test as compared to culture method. Out of 83 isolates, serogroup B was found to be most predominant (74.69%) as compared to serogroup E (21.68%) and thus confirms our earlier findings [12,13]. Similarly, serogroup B was encountered as the predominant serogroup in our neighbouring country Bhutan [17]. However, serogroup E was recorded as the most frequent among Nepalese isolates [19]; it is relatively uncommon in Australia [20], UK [21] and USA [22]. In addition, we also isolated serogroup I for the first time here. However, its prevalence was found to be least as compared to serogroups B and E. This is in agreement with the findings of Moore et al [2] who also reported the presence of serogroup I of D. nodosus in sheep with a similar prevalent rate (3.5%) in England and Wales. Mixed infection with two different serogroups detected in the present study corroborates with the findings of earlier investigators [2]. The detection of serogroup B in majority of the samples and serogroup E in good number of samples suggests that strains of serogroups B and E could be an appropriate candidate for the development of an effective vaccine. However, further studies in this direction covering more number of flocks and geographical area will precisely determine the formulation of an effective vaccine against the disease in this region. In the present study, 72.28% isolates carried the intA gene thus were considered as virulent strains and this explains the virulent nature and severity of lesions of footrot in the affected animals. We failed to isolate intA positive D. nodosus from 27.72% of the samples.
This could be due to that the sheep with virulent footrot may carry benign strains [8]. In conclusion virulent strains of D. nodosus belonging to at least three different serogroups with predominant prevalence of serogroups B and E are causing virulent footrot in sheep population in Kashmir (India). Our findings also suggest the formulation of a bivalent vaccine from serogroups B and E against the menace in the state. Acknowledgements The authors are highly thankful to Dr. O.P. Dhungyel, Faculty of Veterinary Medicine, University of Sydney, Cambden, NSW 2570, Australia, for the supply of serogroup specific positive control DNA. References [1] Stewart D. Footrot in sheep. In: Egerton JR, Yong WK, Riffkin GG, editors. Footrot and foot abscesses of ruminants. Boca Raton, Florida: CRC Press; 1989. p. 5–45. [2] Moore LJ, Wassink GJ, Green LE, Grogono-Thomas R. The detection and characterization of Dichelobacter nodosus from cases of ovine footrot in England and Wales. Vet Microbiol 2005;108:57–67. [3] Claxton PD. Serogrouping of Bacteroides nodosus isolates. In: Stewart DJ, Peterson JE, McKern NM, Emery DL, editors. Footrot in ruminants. Proceedings of a workshop, Melbourne, 1985. Glebe, Sydney, N.S.W, Australia: CSIRO, Division of Animal Health and Australian Wool Corporation; 1986. p. 131–134. [4] Ghimire SC, Egerton JR, Dhungyel OP, Joshi HD. Identification and characterization of serogroup M among Nepalese isolates of Dichelobacter nodosus, the transmitting agent of footrot in small ruminants. Vet Microbiol 1998;62: 217–233. [5] La Fontaine SL, Egerton JR, Rood JI. Detection of Dichelobacter nodosus using species-specific oligonucleotides as PCR primers. Vet Microbiol 1993;35: 101–117. [6] Dhungyel OP, Whittington RJ, Egerton JR. Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination. Mol Cell Probes 2002;16:285–296. [7] Palmer MA. A gelatin test to detect activity and stability of proteases produced by Dichelobacter (Bacteroides) nodosus. Vet Microbiol 1993;36:113–122. [8] Cheetham BF, Tanjung LR, Sutherland M, Druitt J, Green G, McFarlane J, et al. Improved diagnosis of virulent ovine footrot using the intA gene. Vet Microbiol 2006;116:166–174. [9] Katz ME, Howarth PM, Young WK, Rifkin GG, Depiazi LJ, Rood JI. Identification of three gene regions associated with virulence in Dichelobacter nodosus, the causative agent of ovine footrot. J Gen Microbiol 1991;137:2117–2124. [10] Rood JI, Howarth PA, Haring V, Yong WK, Liu D, Palmer MA, et al. Comparison of gene probe and conventional methods for the diagnosis of ovine footrot. Vet Microbiol 1996;52:127–142. [11] Liu D, Webber J. A polymerase chain reaction assay for improved determination of virulence of Dichelobacter nodosus, the specific causative pathogen for ovine footrot. Vet Microbiol 1995;43:197–207. [12] Wani SA, Samanta I, Buchh AS, Bhat MA. Molecular detection and characterization of Dichelobacter nodosus in ovine footrot in India. Mol Cell Probes 2004;18:289–291. [13] Wani SA, Samanta I, Shah SS. Isolation and characterization of Dichelobacter nodosus from ovine and caprine footrot in Kashmir, India. Res Vet Sci 2007;83:141–144. [14] Egerton JR, Roberts DS. Vaccination against ovine footrot. J Comp Pathol 1971;81:179–185. [15] Thorley CM. A simplified method for the isolation of Bacteroides nodosus from ovine footrot and studies on its colony morphology and serology. J Appl Bacteriol 1976;40:301–309. [16] Egerton JR, Ghimire SC, Dhungyel OP, Shrestha HK, Joshi HD, Joshi BR, et al. Eradication of virulent footrot from sheep and goats in an endemic area of Nepal and an evaluation of specific vaccination. Vet Rec 2002;151:290–295. [17] Gurung RB, Dhungyel OP, Tshering P, Egerton JR. The use of an autogenous Dichelobacter nodosus vaccine to eliminate clinical signs of virulent footrot in a sheep flock in Bhutan. Vet J 2006;172:356–363. [18] Cagatay IT, Hickford JGH. Update on ovine footrot in New Zealand: isolation, identification and characterization of Dichelobacter nodosus strains. Vet Microbiol 2005;111:171–180. [19] Ghimire SC, Egerton JR, Dhungyel OP. Characterization of Dichelobacter nodosus isolated from footrot in sheep and goat in Nepal. Small Rumin Res 1996;23:59–67. [20] Claxton PD, Ribeiro LA, Egerton JR. Classification of Bacteroides nodosus by agglutination test. Aust Vet J 1983;70:123–126. [21] Hindmarsh F, Fraser J. Serogrouping of Bacteroides nodosus isolated from ovine footrot in Britain. Vet Rec 1985;116:187–188. [22] Gradin JL, Sonn AE, Petrovska L. Serogrouping of Bacteroides nodosus isolates from 62 sources in the United States. Am J Vet Res 1993;54:1069–1073.