AN OUTBREAK OF HAEMORRHAGIC SEPTICAEMIA IN THE NAVITHANVELI VETERINARY RANGE IN AMPARA DISTRICT , SRI LANKA

INTRODUCTION disease (Benkirane and De Alwis, 2002). Vaccination using inactivated culture has been used to overcome the Hae mor rha gic Sep tic aem ia (HS ) is a fat al disease in many countries and in Sri Lanka. Vaccination septicaemic disease of bovines where water buffaloes must initiate at 4 months and continued annually in Sri are more susceptible than cattle (Bain et al., 1982). It is Lanka (De Alwis., 1999) caused by P. multocida, a Gram negative cocobacilli HS is seen in tropical countries in Africa, South and which colonize the nasopharynx (Wijewardana, 1992; South East Asia, Southern Europe and Middle East. HS Tabatabaei et al., 2007). Specific serotypes of P. is a devastating alarming problem in cattle and multocida B: 2 and E: 2 cause HS in Asia and Africa buffaloes in Asia because of economic loss and crop respectively (Benkirane, 2002; Carter, 1955). P. redundant (De Alwis, 1999). HS was first reported in multocida can survive for some period in soil or water 1911 in Sri Lanka but epidemics occurred in mid 1950s (Bain et al., 1982) where viability will be reduced after with 5000 deaths of cattle and buffaloes. However, 2-3 week. Direct contact with infected animals and vaccination started in 1957 but annual vaccination was fomites are the main sources of transmission of the inaugurated in 1984 in Sri Lanka (De Alwis, 1992). causative organism and bovines are infected due to Consequently in the epidemics reported in early 1980s ingestion of contaminated pasture or water. In endemic and 1990s, the mortality had declined progressively areas, up to 5% of cattle and water buffalo may act as (FAO, 1991; De Alwis, 1999). De Alwis reported HS carriers (De Alwis., 1992) and harbour organism in their was endemic in 13 districts covering two-third of the nasopharynx. Stress, poor nutrition, close herd, wet island during 1980s. Sri Lanka has self-declared HS on th humid weather leads to spreading of the disease to 12 December 2012 following massive national efforts buffaloes and cattle during paddy cultivation in the through passive, active and serological surveillance north east monsoon. Furthermore moist environment according to OIE terrestrial animal health code (OIE, facilitate the longer survival of the organism outside the 2012). This article describe the reoccurrence of HS in host (De Alwis, 1999) water buffaloes and cattle in Navithanveli veterinary Most cases in cattle and buffaloes are acute or per range in Ampara District probably the first re-emerging acute with signs of depression, fever, salivation and a episode in the Ampara district. serous nasal discharge. Furthermore they develop oedematous swellings in the pharyngeal region which MATERIALS AND METHODS spread to the ventral cervical region and brisket, causing hyperaemic mucous membranes and Outbreak location respiratory distress. Moreover, animals collapse and die Navithanveli is situated in the Ampara District 6-24 hours after appearence of clinical signs (De Alwis bordering from East and West by Kalmunai (Kittange et al., 1992). Mortality is nearly 100% unless the animal Lake) and Uhana range, South and North by is treated very early with antibiotic during the pyrexic Samanthurai and Thumberkerny (Batticalo District). stage. Sanitary measures and vaccination should be Navitanveli experience a long dry period which extends carried out in-order to minimize the occurrence of the from February to November. Main occupation of the SUMMARY: Haemorrhagic septicaemia (HS) is a major diseases of bovines in Sri Lanka before 2005. The disease is endemic in dry zone where clinical incidences occur with the onset of monsoon. An outbreak of HS occurred in twenty 1-3 year old local buffaloes of which 15 died, in mid-October 2016 in Navithanveli veterinary range in Ampara District. The disease continued and another 13 buffaloes and 3 cattle died. Clinical and necropsy findings and laboratory identification confirmed that the causative organism was Pasrerella multocida serotype B:2. Outbreak was controlled with treatment and vaccination of all susceptible animals. This is the first report of the re-emergence of P. multocida after 2000 in Ampara district. DOI: http://doi.org/10.4038/slvj.v64i2.22

th humid weather leads to spreading of the disease to 12 December 2012 following massive national efforts buffaloes and cattle during paddy cultivation in the through passive, active and serological surveillance north east monsoon.Furthermore moist environment according to OIE terrestrial animal health code (OIE, facilitate the longer survival of the organism outside the 2012).This article describe the reoccurrence of HS in host (De Alwis, 1999) water buffaloes and cattle in Navithanveli veterinary Most cases in cattle and buffaloes are acute or per range in Ampara District probably the first re-emerging acute with signs of depression, fever, salivation and a episode in the Ampara district.serous nasal discharge.Furthermore they develop oedematous swellings in the pharyngeal region which MATERIALS AND METHODS spread to the ventral cervical region and brisket, causing hyperaemic mucous membranes and Outbreak location respiratory distress.Moreover, animals collapse and die Navithanveli is situated in the Ampara District 6-24 hours after appearence of clinical signs (De Alwis bordering from East and West by Kalmunai (Kittange et al., 1992).Mortality is nearly 100% unless the animal Lake) and Uhana range, South and North by is treated very early with antibiotic during the pyrexic Samanthurai and Thumberkerny (Batticalo District).stage.Sanitary measures and vaccination should be Navitanveli experience a long dry period which extends carried out in-order to minimize the occurrence of the from February to November.Main occupation of the people is paddy cultivation which depends on North east the QIAGEN multiplex PCR kit.Primer sequences monsoon extending from November to February.The (Townsend et al., 1998) were used for P. multocida first incidence occurred in the Chavalakada Grama specific KMT1T7: 5'-ATC-CGC-TAT-TTA-CCC-AGT-Niladhari (GN) area adjacent to the Kittange lake.
3' RGPMA6: 5'-ATT-GTT-TGC-GAT-AGT-CCG-TAG-A-3' and HS-causing type-B-specific KTT72: 5'-AGG-History and clinical signs CTC-GTT-TGG-ATT-ATG-AAG-3' KTSP61: 5'-ATC-First occurrence was reported with the sudden death of CGC -TAA -CA C-A CT-C TC-3' des cri bed in OIE 15 local buffaloes including eight females and seven terrestrial manual 2012.Samples were cultured aerobically on 5% blood agar Necropsy findings and on Mac Conkey agar plates and incubated at 37° C for Necropsy examination of three buffaloes revealed 24 hours.Positive cultures were subjected to biochemical congested lungs, petechial haemorrhages on cardiac tests (Indole, Oxidase and Nitrate) and mouse inoculation parenchyma, sub-epicardia l adipose tissue, serosal (sub cuta neou s or intr a-pe rito neal rout e).Furt her surface of abdominal organs, and blood tinged fluid in the confirmation was done by serotyping, agglutination test body cavities (Figure 3).with serum raised against Pasturella multocida B: 2 followed by agar gel precipitation test.

Laboratory findings Smooth, greyish glistening translucent colonies on Serogroup identification by Polymerase Chain Reaction
blood agar revealed characteristics similar to that of P. multocida colonies.Leishman stain indicated Gram DNA extraction negative bipolar short coccobacilli.There was no growth Genomic DNA isolation from samples were performed on MacConkey agar.Indole, oxidase and nitrate using the tissue sample protocol of the QIA amp DNA biochemical tests were positive.Further, in mouse (QIAGEN, Germany) purification mini kit according to inoculation test death occurred after 18 hours and gram the manufacturer's instructions with a loop full of cells negative bipolar coccobacilli were isolated from heart (culture) suspended in 200µl of distilled water.Samples blood.Serotyping revealed that the isolate agglutinated were centrifuged for 5 minutes at 3000 rpm.The with specific serogroup P. multocida type B: 2. supernatant was removed completely and discarded.
Pathogenic strain was confirmed as P. multocida serotype B by multiplex PCR with the amplification product sizes Mulitplex PCR using HS causing type B of 457bp, 620bp and 720bp respectively (Figure 4).The multiplex PCR was performed in a Gene Amp PCR Conventional bacteriological test, serotyping and system 9700 (Applied Biosystems) thermal cycler using molecular findings confirmed the isolate is P. multocida time all the susceptible animals in herds were vaccinated observations made by De Alwis (1992).Furthermore, with inactivated P. multocida combined with alum paddocking together at night, using common grazing land adjuvent as a control measure.Following early treatment, and water resource are involved in spreading of the 24 buffaloes and 21 cattle successfully recovered but 4 disease to adjacent buffaloes and cattle herds (Chavalaka, buffaloes died despite treatment.Dead carcases were Annmalaia 01 and Annamalai 02 GN division) during this deeply buried and infected areas were burnt with straw.
outbreak.Furthermore routine preventive vaccination was initiated Under field conditions, HS is usually diagnosed on the against HS in Ampara District to prevent further basis of clinical signs and symptoms (Khan et al., 2006).occurrence of the disease.
Early treatment depends on the early diagnosis of the disease (De Alwis, 1999).Respiratory distress, dyspnoea, DISCUSSION high temperature, reduced appetite, restlessness and hypersalivation were present in affected buffaloes similar Outbreaks of HS usually occur in many Asian and to that observed by Sheikh et al., (1996).African countries resulting in high mortality and HS is effectively treated by broad spectrum antibiotics.morbidity (Bain et al., 1982;De Alwis, 1992).According According to Benkirane and De Alwis (2002), animals to this outbreak, HS occurred with the onset of Northeast can be recovered if they have been treated in the very monsoon in Navithanveli veterinary range from October early stage of the disease.Penicillins are the drug of to February.Hot and humid climatic condition is a major choice of HS because the causative organism is sensitive contributory factor in this outbreak of HS where high to this â-lactam antibiotics (Kristinsson and Adam, 2007; environmental temperature facilitates the multiplication Pedersen et al., 2009).This isolate was sensitive for wide of the bacteria outside the host (Hajikolaei et al., 2008).range of antibiotics however, tetracycline was used for This is similar to the observations made by De Alwis treatment and 45 animals who developed clinical signs (1992).Majority of the affected animals in this outbreak recovered after treatment.Nevertheless four animals died were buffaloes where 25 died and among them 20 were due to severity of the infection.Early intervention from the same herd.These affected buffaloes were mainly together with treatment and vaccination overcome the reared for paddy cultivation during north east monsoon in outbreak of HS within short time period.the area where they freely roam in the lands.Harvesting, P. multocida produces endotoxins which causes the lack of food, overcrowding, and poor hygiene are stress toxaemia (Horadagoda et al., 2001;Zafar et al., 2010).factors to the animals that facilitate harbouring of During necropsy, it was observed in the present outbreak organisms in their nasopharynx similar to the that haemorrhages of abdominal organs, heart, congested th PCR reaction consisted with 1 × PCR buffer, 200 µM males on 17 October 2016 with the onset of North east each deoxynucleotide triphosphate (dNTP), 2 mM monsoon.However, there were five local buffaloes MgCl , 3.2 pmol of each primer and 1 µl Taq DNA showing clinical signs of depression, severe respiratory 2 distress, submandibular, ventral cervical and brisket polymerase.PCR was done in a final volume of 25µl with 0 oedema, hyperaemic mucous membranes, fever (106 F) initial denaturing at 95°C for 5 minutes, 30 cycles of and hyper salivation (Figure 2).All the affected animals denaturing at 95°C for 1 minute, annealing at 55°C for 1 were within the age group of 1-3 years.Furthermore the minute and extension 72°C for 1 minute, with a final 0 outbreak continued with sudden death of 5 buffaloes in extension at 72 C for 7 minutes.Distilled water adjacent herds (Chavalakada, Annmalai 01 and Annmalai (QIAGEN) was used as a negative control.A volume of 5 02) where 28 showed clinical signs.Moreover the µl of each sample was electrophoresed on a 2% agarose outbreak spread to the cattle herds in Chavalakada and gel in 1 × Tris-acetate running buffer (TAE) at 4 V/cm for Annmalai 01, with 24 clinically affected animals of which 1 hour.The gel is stained with 1% ethidium bromide and 3 died in a short period of time.The condition in cattle was DNA fragments are viewed by UV trans illumination.less severe than buffaloes as they have shown only hyperaemic mucous membrane, hyperthermia and Antimicrobial susceptibility test respiratory distress.Antimicrobial susceptibility test was carried out by the disc diffusion method according to the manuals of clinical Laboratory Investigation laboratory standard institute.Cephalexin (30µg), Amoxicillin clavulunate (augmentin) (30µg), Bacteriological analysis Tetracycline (30µg), Cloxacillin (10µg) and Penicillin Post mortem on 3 buffaloes and one cattle was (10µg), Erythromycin (5µg), Neomycin (30µg) and th th conducted in 18 and 25 October.Samples of heart Enrofloxacin (5µg) discs (HIMEDIA, India) were used blood, tissue samples of lung, liver and spleen were for the test.collected and dispatched in ice to the Central Veterinary Investigation Centre at Veterinary Research Institute, RESULTS AND DISCUSSION Gannoruwa.
Figure 1.Geogrophical distribution of Navithanveli veterinary Divisional secretarial division (Blue colour indicate outbreak locations) Source; Navithanveli Administrative map