1 In ternationa l Scholars Journa ls African Journal of Pig Farming ISSN: 2375-0731 Vol. 9 (1), pp. 001-006, January, 2021. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper Seasonal emergence of swine erysipelas in hilly state Nagaland, Northeast India Nagendra Nath Barman1*, Debojyoti Borkotoky2, Biswajyoti Borah1, Anjan Jyoti Nath3, Papiya Das1 and Durlav Prasad Borah1 1 Department of Microbiology, College of Veterinary Science, Assam Agricultural University, Khanapara campus, Guwahati-781022, Assam, India. 2 Subject Matter Specialist (Animal Science), KVK Phek, ICAR-NRC on Mithun, Nagaland-797007, India. 3 Department of Microbiology, Lakhimpur College of Veterinary Science, Assam Agricultural University, Joyhing, North Lakhimpur- 787 051, Assam, India. *Corresponding author. E-mail: nnbarman@gmail.com. Accepted 20 September, 2019 Seasonal outbreaks of swine erysipelas have been reported in back yard pig farms in the Phek district of Nagaland, India. The alpha haemolytic isolate of Erysipelothrix rhusiopathiae was recovered on blood agar from the clinical samples. The organisms were confirmed microscopically, biochemical analysis as well as by polymerase chain reaction (PCR) amplification of 16S rRNA gene and sequence analysis. These Nagaland isolates (KT160358, KT160359) were closely related to the type spp. E. rhusiopathiae in phylogenetic analysis and forms the same clad with Chineese isolates of swine and murine origin indicating an epidemiological link. The isolates were found to be most sensitive to oxytetracycline and responded to treatment. Swine erysipelas occurred in Phek district in a season due to sudden change of weather and temperature. Pigs exposed to such predisposing factors probably favoured to propagation of already persisted organisms in pigs. This is the first confirmed case of E. rhusiopathiae infection from the NE states of Nagaland, India. Key words: Swine erysipelas, Erysipel othri x rhusiopathiae, pig, polymerase chain reaction (PCR) Nagaland, India, Oxytetracycline. INTRODUCTION Erysipelothrix rhusiopathiae, belonging to the family Erysipelotrichac eae, is a non-motile, Gram-positive, non- sporulating, non-acid-fast organism distributed worldwide affecting wide variety of vertebrate and invertebrate species including man (Reboli and Farrar, 1989). Organisms in many occasions harbour by pigs in lymph nodes and shed along with feces, urine, saliva and nasal secretions (Lee et al., 2011). Affected pigs manifest the disease as (i) acute septic form, (ii) subacute urticarial form marked by reddis h-purple rhomboid spots or "diamonds" in the skin, (iii) joint or arthritic form, and (iv) chronic cardiac form (endocarditis) (Reboli and Farrar, 1989).Various predisposing factors, change of environmental conditions and parasitic infestation lead to 2 reappearance of s wine erysipelothrix (SE) infection in that population. Seasonal outbreaks of swine erysipelas were investigated in Phek district of Nagaland, India during 2013-2015. MATERIALS AND METHODS Outbreaks of Sw ine erysipelas – samples collection The disease of sw ine erysipelas w as reported from Porba village (altitude 1985 MSL), District Phek of Nagaland during the summer rainy season from 2013 to 2015. The village had a pig population of 552 cross bred and 200 local doom pigs as per the livestoc k census report 2012 (GOI, 2012). Far mers keep pigs as a bac k yard s mall unit mainly for meat purpose and fed them on household as w ell as hotel w aste. In every rainy summer season ( May – July) there w as disease outbreaks in pigs. Affected animals (150) clinically w ere anorexic and recumbent w ith high fever (106°F) for 2-3 days. Some animals developed erythematous patches. Postmortem examination revealed haemorrhages in intestine, congestion of liver, spleen and kidney. During the course of investigation 3 more animals died w ithin 12 days. All affected pigs w ere also reported to be infested w ith pig louse Haemat opinus suis. Clinical sample like sw abs from the w ound and tissue biopsy from the affected areas were collected in sterile containers for isolation of the causative organis m. Samples also included sloughed off tissues and biopsy samples preserved in 10% formalin for histopathological study . Virological investigation Tissue samples w ere processed for demonstration of classical sw ine fever using single step Reverse transcription poly merase chain reaction ( RT- PCR) (Hoffmann et al., 2005) and sw ine pox as per the method of Medaglia et al. (2011). Identification and antibiotic activity of Erysipelothrix rhusiopathiae The sw ab samples ( N=51) from the affected areas w ere inoculated into nutrient broth and incubated aerobically at 37°C for 48 h. Sub-culturing w as done on blood agar plates, incubated at 37˚ C in presence of 5% CO2 for 24 h. Colony mor phologies w ere studied. Gram’s staining and biochemical analysis w ere done to confirm the organis m. A panel of antibiotic discs containing amikacin (30 µg), amoxicillin (30 µg), ampicillin (10 µg), cefotaxime (30 µg), chloramphenicol (30 µg), c iprofloxacin (5 µg), cloxac illin (30 µg), co-trimoxazole (25 µg), enrofloxacin (5 µg), gentamicin (10 µg), neomycin (30 µg), norfloxacin (10 µg), oxytetracycline (30 µg), streptomycin (25 µg) and tetracycline (30 µg) w as used to study the antibiotic sensitivity patter n of the isolates. The zone of inhibition was measured, recorded and interpreted according to the clinical and Laboratory Standards Institute criteria (CLIS-MIC). Detection of nucleic acid of Erysipelothrix rhusiopathiae and sequencing For detection of E. rhusiopathiae nucleic acid tissue samples w ere processed using DNA Sure® Tissue Mini Kit ( Nucleo-pore, cat.#NP-61305) and poly merase chain reaction ( PCR) amplification for 16S r RNA gene pr imers ( MO101 F AGATGCCA TAGAAACTGGTA and M0102 R CTGTATCCGCCA TAA CTA) of E. rhusiopathiae ( Makino et al., 1994) w as used. The PCR conditions w ere optimized w ith a final volume of 25 µl at 94°C for 5 min follow ed by 30 cycle of 94°C for 30 s, 54˚C for 2 min and 72˚C for 45 s and final extension w as carried out at 72°C for 5 min. In PCR r eaction 28 ng/ µl total genomic DNA w as taken along w ith positive and negative control. Pr oducts of PCR w ere visualized in 2% agarose gel electrophoresis under Geldoc ( Kodak, USA). Further PCR products w ere purified by QIAquick PCR purification kit protocol and sequenced. Analysis of gene sequence and phylogenetic studies on Erysipelothrix rhusiopathiae To deter mine the relationship of the Nagaland isolates of E. rhusiopathiae w ith other isolates of this species, 16S r RNA gene was amplified and sequenced (GenBank accession number KT160358 and KT160359). Sequence identity at nucleotide level was deter mined by Clustal W method of Meg-Align program in DNA STA R pac kage ( DNASTA R Inc., USA). The phylogenetic tree was constructed using other 16S r RNA gene sequences available at NCBI (viz. strain ZYL(KF811052.1), Isolate EU188793.1, strain:KG- BB1(AB055909.1), strain Fujisaw a(NR_074878.1), Isolate DQ462571.1, strain: A TCC 19414(AB055905.1), strain JPB251209S( HM569359.1), Chiba9393( EF494748.1), sp. T127_5( JQ739693.1), sp. LV19(KJ670316.1), strain Er.GXLC- 1(KP063151.1), inopinata strain 143-02 ( inopinata strain 143-02) strain: JCM 8534(LC019778.1) and strain Er.GXBY -1(KP063149.1). Sequences w ere aligned by ClustalX version 2.1 (www .clustal.org), and the concatenated alignments w ere used for phylogeny inference ( MEGA5; www .megasoftw are.net) opting for the Maximum parsimony and Poisson correction. Computed r eplicates for bootstrap support w as done and values w ere observed. Histopathology For malin fixed tissues w ere processed as per standard protocol for histopathological studies. Sections of 4-5 micron thickness w ere stained routinely w ith Haematoxylin and Eos in stain and observed under oil immersion objective of a low pow er light microscope. RESULTS AND DISCUSSION Tissue samples processed for detection of classical swine fever virus and swine pox virus were confirmed as negative for both viral agents. However, bacteriological investigation demonstrated association of E. rhusiopathiae infection in affected (5) as well as in dead (2) pigs. Prevalence of erysipelothrix in many animals, mostly in pigs and birds has been reported throughout the world including from India (Shank ar et al., 2009; Arora et al., 2011). But there was a single report on swine erysipelothrix (SE) from Meghalaya (Das et al., 2014). North Eastern Region has the highest pig population of the country. Diverse geographical locations, varied climatic situations and frequent movement of pigs favour for spread of the disease through carrier pigs (Leslie et al., 2015). Present report is a thorough investigation on swine erysipelothrix occurred seasonally at Nagaland, another NE state of India. Clinical signs A total of 150 pigs during 2013-15 exhibited clinically high fever (105-107 0 F), anorexia, firm faeces, and animals 3 Figure 1. Character istic lesions of sloughed off skin from the cases of E. rhusiopathiae infected pigs. lying down. Cutaneous lesions appeared after 5-7 days of illness initiating with erythematous patches followed by papules of 4-5 mm diameter, dark purple, raised, firm to touch giving square to rhom boid shape in entire body. No vesicular stage was noticed. Although scabs developed in the entire body, prominent lesions were identified on head, face back, belly, limbs, tail and on ear (Figure 1). In unattended cases scab lesions were sloughed off within 17-22 days leaving a large ulcerated area. Thes e typical cutaneous lesions have been mostly seen in grower animals. In affected pigs it appears that cutaneous lesions were developed following acute stages of illness. Considering the pathogenesis of E. rhusiopathiae, organisms gain access to the body, probably through the tonsils or other lymphoid tissue of the digestive tract and spread throughout the body. The bacteria produce neuraminidas e, an enzyme that cleaves mucopolysaccharides in cell walls which may mediate the wides pread vascular damage that accompanies SE. Vascular damage leads to thrombosis and interference with microcirculation in capillaries and venules at many sites. Classic cutaneous rhomboid urticaria (diamond skin) occurs in a percentage of pigs shortly after the acute febrile stages. In younger pigs with acute erysipelas, signs are similar, with cyanosis of extremities, ears and snouts pronounced and urticaria less common (Anonymous, 2016). Seasonal occurrence of swine erysipelas in Nagaland justifies the stress due to sudden change of temperature. As stated by Amanda (2012)that stress factors such as overstocking, mixing pigs after weaning, and sudden changes in temperature can trigger clinical erysipelas. Isolation and identification of organism On blood agar the organism produced small, circular and transparent α- hemolytic colonies with a smooth glistening surface and edge. Biochemically all the isolates were catalase, oxidase and urease negative, produces H2S and ferments glucose and lactose. All 7 isolates were identified as E. rhusiopathiae based on the cultural, morphological and biochemical characteristics. The Nagaland isolates were sensitive in vitro to oxytetracycline, tetracycline, ampicillin, amoxicillin and cloxacillin; moderately sensitive to streptomycin, enrofloxacin, amikacin, co-trimoxazole, cefotaxime and ciprofloxacin; and resistant to gentamicin and norfloxacin, chloramphenicol and neomycin. Based on the antibiogram, survived ailing pigs were treated with oxytertacycline at 10 mg/kg body weight, intramuscularly for one week. Eight out of 13 treated animals responded promptly and recovered. Treatment of swine erysipelas cases with penicillin (Shankar et al., 2009) or other penicillin group of drugs such as the combination of amoxycillin and cloxacillin (Das et al., 2014) have been frequently reported. Molecular confirmation and characterization Tissue samples were negative for Swine fever virus and pox virus in PCR. A total of seven E. rhusiopathiae isolates recovered from tissue samples were subjected for PCR amplification using 16s rRNA gene s pecific primer set. All isolates were found to be positive for E. rhusiopathiae with amplification products of 407bp. 4 500 bp 407 bp 100 bp Figure 2. 2% agarose gel show ing PCR Products. Here, L1- 100 bp marker, L2 NTC, L3& 4 PCR products. Figure 3. Phylogenetic tree of Nagaland isolates of E. rhusiopathiae (KT160358, KT160359 as compared to the ATCC 19414 type strain and other isolates. (Figure 2). Two Nagaland isolates KT160358 and KT160359 were sequenced and showed high sequence identity with the sequences of other E. rhusiopathiae available in the GenBank database. The Nagaland isolates (accession numbers KT160358, KT160359) were in the same clad (Figure 3) along with other strains originated from China. Phylogeny based on the nucleotide and amino acid sequences of the viruses provides a better understanding of the molecular epidemiology of the isolates. The state 5 Figure 4. Histopathology of skin biopsy sample, H&E, 100X. Congestion and presence of microthrombi in the capillar ies of the der mis along w ith infiltration of lymphoid cells and fibroblast w ere consistent finding. Nagaland shares international boundaries with My anmar and having territorial link with other neighboring international countries like China, Bhutan and Bangladesh. The Chinese strains used in the construction of phylogenetic tree viz. swine isolates KPO 63150.1, strain Er.GXBY-1(KP063149.1), strain Er.GXLC-1(KP063151.1) and murine isolate DQ4625711are forming same clad with Nagaland isolates ( KT160358 and KT160359) indicated an epidemiological link. Movements of animals and animal products might spread the infection to this locality. Histopathology Histopathologic alteration of affected skin showed extensive damage to the capillaries and venules of dermis with infiltration of lymphoid c ells and fibroblasts. Congestion and pres ence of microthrombi in the capillaries were consistent finding in all skin biopsy samples investigated in the present study (Figure 4). Similar observations were also recorded by Shankar et al. (2009). Cultural characteristics, molecular confirmation and histopathological changes conclusively proved that swine erysipelas is prevailing in Nagaland. Pigs harbouring the infection manifest clinically at sudden change of climate. It is estimated that 30–50% of healthy swine harbour the organism in their tonsils and other lymphoid tissues (Stephenson and Berman, 1978). Again, trans border movement of pigs and their products could facilitate spreading of SE in this locality. Further study on prevalence of swine erysipelas and identification of carrier pigs can provide actual guidelines to control the disease in this part of India. Conclusion Unorganized pig farms in the Phek district of Nagaland experienced high mortality of grower pigs during rainy- summer season. A detail isolation and molecular investigation confirmed the association of swine erysipelas in this part of north eastern region of India. 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