Soil-borne Septicaemic Colibacillosis in Neonatal Lambs: Salient Observations

In sheep husbandry, neonatal lamb (<4 weeks) mortality is a cause of grave concern as it can drastically diminish the farm returns. Enteric infections are quite commonly observed in week-old lambs. Enteric colibacillosis and septicaemic colibacillosis are the two most common forms of illness and E. coli accounted for 65% of septicaemic cases in lambs and major cause of diarrhoea in young calves (Sharma and Joshi, 2020). Septicaemic colibacillosis is highly fatal in young pre-ruminants and therapeutic options are limited even if they are exercised in time. In the semi-arid tropical geography of Rajasthan, India, an outbreak of septicaemic colibacillosis occurred in three sheep flocks. Perusal of literature revealed that there is dearth of epidemiological information on septicaemic colibacillosis. Hence, in this article, the salient epidemiological, pathological observations and response to management are reported herewith.

Location and flock particulars
 
The flocks were located in the ICAR-Central Sheep and Wool Research Institute, Avikanagar, Rajasthan, India (Geocoordinates: 75^o 28’E and 26^o 17’N) and mean annual rainfall had been around 500 mm (Swarnkar and Singh, 2011). Flock A had only native Malpura breed of sheep and flock B had both Malpura and crossbred (Avikalin- 50% Malpura and 50% Rambouillet) sheep whereas flock C had only crossbred (Garole and Malpura crosses) sheep. All three flocks were maintained in semi-intensive system and standard feeding practices were followed along with grazing. Deworming, vaccination and dipping were carried out as per planned flock health programme.
 
Clinical examination and necropsy
 
During February 2019, sudden death of neonatal lambs was observed. Information pertaining to the onset of epidemic and preceding management activities were collected. As soon as possible, all ailing and dead lambs were subjected for clinical and post-mortem examination, respectively. Tissue samples like liver, lung, heart and kidney were collected and histopathological examination was carried out as per standard procedure (Culling, 1968). While performing the necropsy, swab samples from the liver, lung, heart and gastrointestinal tract were also collected and subjected for standard bacteriological examination and antibiogram by Kirby-Bauer disc diffusion method.
 
Virulence characterization of the E. coli by PCR
 
Genomic DNA was extracted from pure cultures of E. coli using Qiagen DNeasy® Blood and Tissue kit, according to manufacturer’s instructions. The eluted genomic DNA was quantified using a Quawell UV-Vis Q5000 spectrophotometer (Quawell Technology Inc., San Jose, CA, USA). After checking the DNA purity on 0.8% agarose (Sigma, USA) gel electrophoresis, they were stored at - 20°C till further use in the PCR assay. The conventionally identified E. coli strains were confirmed by 16S rRNA PCR, PCR product sequencing and NCBI blast analysis.  The presence of some selected virulence genes like shiga toxin (stx2) and intimin (eae) were studied by PCR. The oligonucleotide primer sequences along with their amplicon sizes are listed in Table 1. All the primers were synthesized from Sigma Aldrich (Bengaluru, India). The condition adopted for their amplification like annealing temperature, primer concentration etc. were standardized and the optimized cycling and reaction conditions were used to screen samples in a thermo cycler (peqSTAR 96 Universal Gradient, peqlab, Germany) As negative control, all components of the reaction mixture without DNA template was included in the PCR. The PCR products were analyzed as described by Sambrook and Russell (2001). For sequencing of the amplicons, standard PCRs were run and PCR products (50 µl) were resolved on agarose gel. The desired sized bands were excised from agarose gel under UV light and were subsequently purified using MinElute® Gel Extraction Kit (Qiagen). The purified products were sequenced (Xcelris Genomics, Ahmedabad, India) and searched with the sequences available in the NCBI database.

Epidemiological observations
 
Inclement conditions like cold, wet and windy weather are the major precipitating factors for colisepticaemia (Sonawane et al., 2012). During this outbreak, the mean maximum and minimum temperatures were 25.4^oC and 12.6^oC, respectively. In the first incidence (Flock A) that occurred in the beginning of February 2019, there were 163 lambs (males 77; females 86).  Number of susceptible lambs in flocks B and C were 68 and 173, respectively. The mean age and birth weight of the dead lambs (n=404) were 11.00±0.49 days and 3.09±0.82 kg for males and 2.94±0.68 kg for females, respectively. Native Malpura lambs (87.5%) were predominantly affected. Lamb mortality of 44.79% (73/163), 20.59% (14/68) and 5.2% (09/173) was observed in flocks A, B and C, respectively. Many a times, E coli, especially shiga toxin-producing E. coli (STEC), organisms are shed by ruminants like sheep and cattle.
 
Clinical and necropsy observations

The septicaemic form of colibacillosis causes per acute death without recognizable premonitory signs (Radostits et al., 2006). In this outbreak, 46.87% lambs were found dead. However, some lambs exhibited tachypnea (>52/min), fever (>104.2oF) and remain isolated from flock. Evidently, respiratory distress had abdominal component. Bacterial lipopolysaccharides (LPS) can bring about cascade of hemodynamic changes in vasculature, particularly pulmonary vessels. This results in acute development of pulmonary edema, mucus nasal discharge and hypovolemia (Shabankare et al., 2015). Lesions like edema, congestion, haemorrhage, blood-tinged froth in trachea (5.21%) and tracheal mucosal hyperemia were characteristic of acute endotoxaemia (Kusiluka and Kambarage, 1996).
 
In all carcasses, rapid disappearance of rigor mortis was seen. Necropsy further revealed severe congestion and enlargement of liver (90.62%), highly congested renal parenchyma to the extent of haemorrhage (84.38%) and severe pulmonary edema and multifocal petichiae to ecchymotic lesions on the lung surface (89.58%). Very importantly, 57.29% of the lambs had soil-mixed ingesta giving a dirty appearance to the abomasal contents. Among the dead lambs, 25% had hyperemia and necrotic spots on abomasal mucosa. Despite many hypotheses, Slabach et al., (2015) reiterated that geophagy, also a type of pica, is a proximate mechanism to self-address the mineral nutritional demand. In the present study, lambs had dirt/soil in their abomasum which suggested that a possible underlying micro-mineral deficiency in the affected flocks. Autolytic changes were rapid in kidneys (Otter and Davies, 2015) as they are highly metabolically active.  Histopathogical findings were engorged pulmonary vasculature, fibrinous exudation in alveoli, mural thickening of hepatic blood vessels and coagulation necrosis of renal tubules.
 
Bacteriological examination and antibiogram
 
A total of 54 bacterial colonies (from 61 sets gastrointestinal swabs, heart blood/swab, lung swabs and liver swabs) were isolated and 45 of them were identified as Escherichia coli, confirming it as an outbreak of septicaemic colibacillosis. Organisms were sensitive to amikacin (68%), cefixime (60%), ceftazidime (64%), imipenem (33%), chloramphenicol (100%), kanamycin (24%), co-trimoxazole (65%), amoxyclav (25%), gentamicin (91%) and nitrofurantoin (91%). A total of 16 (36%) isolates were resistant to ceftazidime (a third generation cephalosporin), which is suggestive of an extended spectrum β-lactamases (ESBL) producer strains. The conventionally identified E. coli strains were tested for the presence of some selected virulence genes like shiga toxin (stx2) and intimin (eae) gene by PCR. The E. coli isolates were detected positive for harboring major virulence factor genes like shiga toxin (stx2; 381 bp) and intimin (eae; 890 bp) gene by PCR (Fig 1 and 2).




 
Epidemic management and response
 
In light of ABST, sulphamethoxazole + trimethoprim was given intramuscularly to all susceptible lambs and thereafter the mortality showed a declining trend. However, eventually, only after the use of probiotics, outbreak could be controlled. On the next week, similar outbreak occurred in flock C where only probiotic (Lactobacillus spp.) was administered for three days. In both the flocks B and C, lamb mortality could be reduced to 20.59 % (14/68) and 5.2% (9/173), respectively. Especially in young pre-ruminants, probiotics exert their action by promoting optimal maturation of ruminal microbiota, increasing the digestive safety at weaning and reducing the pathogen colonization in gut (Chaucheyras-Durand and Durand, 2010; Dar et al., 2018).

Albeit conventional antibiotic therapy, this outbreak caused heavy mortality in lambs. Ingestion of faeces-contaminated soil by lambs could have resulted in this outbreak. However, it is concluded that climatic factors play vital role in precipitating soil-borne STEC infection. It can be inferred that the use of probiotics could be an effective preventive strategy against soil-borne septicaemic colibacillosis.

The authors are grateful for the support rendered by Director, ICAR-CSWRI and ICAR-All India Network Project on Neonatal Mortality in Farm Animals for providing financial assistance for this study.