Occurrence of Multi-drug Resistant Avian Salmonellae in Commercial Poultry

Human are progressively becoming easy targets to the global health challenges, such as emerging and re-emerging infectious diseases as shown by the COVID-19 pandemic, antimicrobial resistance (AMR) and the escalating numbers of non-communicable diseases (Amuasi et al., 2020). Modernization of livestock farms and globalization of bird breeding trade also helps in transboundary spreading of food-borne bacteria such as Salmonella (Chakraborty et al., 2020). In recent years, problems related to Salmonella have increased significantly due to emergence of multi-drug resistant Salmonella. Increment in antimicrobial resistance interferes with the prevention and control of such organisms and represents a danger to public health (Diab et al., 2019).

Salmonella infection is one of the most important bacterial diseases in poultry causing heavy economic loss through mortality and reduced production (Haider et al., 2004). There are relatively fewer number of reports of salmonellosis from India despite its high prevalence, which can be attributed to limited diagnostic facilities under field conditions and underreporting (Rajagopal and Mini, 2013). The contaminated faecal samples from the poultry sheds and cages can cause contamination of eggs and later chicks and hence control of bacterial infections within the poultry sheds becomes challenging (Tiwari et al., 2021). Salmonella sp. detection in faecal samples is crucial not only for determining the aetiology but it can also aid in the elimination of the illness at the farm level (Hassan et al., 2020).

Keeping the following facts in mind, the present study was conducted to determine the presence of salmonellosis in poultry and their MDR pattern in Jabalpur region of Madhya Pradesh.

Sampling at poultry farms
 
The sample collection was conducted from July 2019 to February 2020 on domestic fowl of all age groups, either sex and breeds. A total of 26 poultry farms including 11-layer farms and 15 broiler farms covering different areas of Jabalpur city were included in the study. Proper data collection was performed and several aspects regarding health status of birds, biosecurity measures and other prevailing management practices.
 
Pathological examination
 
Examination of carcasses of birds (189) received at Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, NDVSU, Jabalpur from various poultry farms, was done for observing gross gastrointestinal lesions associated with salmonellosis. For histopathology, the formalin fixed tissues of liver, spleen and intestine from birds found positive for Salmonella following standard procedure (Slaoui and Fiette, 2011).
 
Isolation of organisms
 
Pooled faecal samples (100 g) were collected from poultry farms in Buffered Peptone Water (BPW) for pre-enrichment and samples were incubated overnight. Liver and intestinal swab samples were also collected in BPW at the time of necropsy. Both samples were then inoculated into Tetrathionate broth and Rappaport Vassiliadis Medium and incubated at 37°C and 42°C respectively for 18-24 hours for selective enrichment of the organism. Organisms from the broth medium were then streaked on to selective media XLD and BGA and incubated at 37°C for overnight for obtaining pure colonies of Salmonella.
 
Identification of organisms
 
Identification of organisms was done based on study of characteristics of colony morphology. Biochemical characterization of bacterial isolates was done using readymade biochemical kit including the motility test for differentiation of motile and non-motile salmonellae.
 
Molecular characterization of Salmonella isolates
 
Direct Polymerase Chain Reaction (PCR) was applied on collected samples using invA-based PCR assay for specific detection of Salmonella as per the protocol developed (Scholz et al., 2001). Species specific PCR was performed for S. Enteritidis targeting ent gene following the protocol (Freitas et al., 2010). The DNA isolation from the confirmed bacterial colonies was performed using chelex resin- based DNA purification protocol as per the method described by earlier workers (Jofre et al., 2005).

The published oligo primers (Table 1) specific to Salmonella and S. Enteritidis targeting invA and ent genes respectively (Galan et al., 1992; Alvarez et al., 2004), were synthesized at Integrated DNA Technology (IDT) Inc. and utilized in present study. The cycling conditions for ent gene-based PCR were carried out according to the protocol of Frietas and co-workers (Freitas et al., 2010) but the band was not clearly visible on the gel. However, better amplified product was obtained after incorporating an initial denaturation for 5 minutes in the reaction programme (Table 2).



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Biofilm assay
 
The biofilm producing ability of the bacteria was determined using crystal violet assay, performed as per the protocol described (Cabarkapa et al., 2015). For ascertainment of difference in biofilm formation, 96-well flat-bottomed polystyrene tissue culture plate was used and quantification of biofilm formation was done through the optical densities obtained. The optical density of the wells was measured at 630 nm using an automated microtiter reader and results were presented as the median value of the six replicates.

Based on the optical densities (OD) produced by bacterial films, strains were classified into the following categories: non-biofilm producers, weak, moderate or strong biofilm producers (Stepanovic et al., 2003).
 
Identification of paratyphoid Salmonella using polyvalent antisera
 
A presumptive identification of motile Salmonella was done by using a commercially available antiserum. A small drop of antiserum (20 μl) was added on a glass slide. A small amount of positive Salmonella culture was then mixed with the antiserum. A positive reaction was visible as clumping on the slide.
 
Antibiotic sensitivity test (ABST)
 
The ABST was conducted by the disc diffusion method using different antimicrobials (Tendencia, 2004). Diameter of zones of inhibition was measured and antibiotics were categorized as susceptible, intermediate or resis­tant. Average of all the isolates was used to analyse the sensitivity of antibiotic toward the isolates.

Based on the above-described methodologies, the following results were recorded:
 
Prevalence of Salmonella at poultry farms
 
All the broiler farms followed deep litter system of housing for birds while cage system was followed in the layer farms. Based on the cultural and morphological characteristics, Salmonella infection was confirmed in 20.0% and 45.45% of the broiler and layer farms, respectively. In post-mortem cases, it was recorded in 1.58% of dead birds.
 
Colony morphology
 
Isolates of Salmonella sp. were observed as 2-3 mm pinkish red colonies with black centre on XLD Agar along with change of colour of media showing reddish appearance as observed earlier (Ranjbar et al., 2020). In BGA, the Salmonella isolates appeared as pinkish white colonies with change of the colour of agar medium from green to pink similar to previous observations of researchers (Sannat et al., 2017).
 
Biochemical characterization of Salmonella isolates
 
The isolates exhibited colour change in the media present in the kit indicative of metabolic changes after 24 hours of incubation. The isolates were found negative for indole, Voges-Prausker test, urease, ONPG and lactose while they were found positive for methyl red, citrate, lysine decarboxylation as well as arabinose and trehalose utilization. However, variable reactions were observed for arginine decarboxylation, maltose and trehalose utilization in our study.

Our findings were supported by the studies conducted by Cox and Williams (1976), Howells et al., (2002), Murinda et al., (2002), Wilson (2004), Markey et al., (2013), Kebede et al., (2016), Khueankhancharoen et al., (2016) and Mali et al., (2019).
 
Motility test
 
Motile Salmonella were identified by the development of dark pink growth and movement of bacteria from the inoculated well no. 1 to well no. 2 in the kit. A positive result of motility for Salmonella sp. was indicated by the spread of the stab line as stated before (Aktar et al., 2016). Out of the suspected Salmonella isolates, 27.27% isolates were found positive for the motility test.
 
Molecular detection of Salmonella by PCR
 
Molecular detection of Salmonella sp. by genus-based PCR
 
Among the isolates confirmed via isolation and identification, 90.0% isolates were found positive for Salmonella. In positive samples, the PCR amplified product of 284 bp for invA gene were clearly visible in the form of bands (Fig 1). Overall prevalence of Salmonella infection was confirmed in 13.33% and 36.36% of broiler and layer farms, respectively. In post-mortem cases, Salmonella was recorded in 20.5% dead birds.


 
Molecular detection of Salmonella enterica Enteritidis
 
With the help of PCR, 11.1% isolate found positive for the motile paratyphoid bacteria Salmonella enterica serovar Enteritidis and the overall prevalence of S. Enteritidis was detected as 3.84% out of the total sampled farms.
 
Sero-grouping of motile isolates using polyvalent antisera
 
Salmonella somatic O poly antisera specific for motile organisms S. Enteritidis and S. Typhimurium were used and among the Salmonella isolates obtained, 18.18% tested positive for Salmonella Enteritidis antisera whereas 9.09% tested positive for Salmonella Typhimurium antisera. A similar prevalence of 9.09% of Salmonella typhimurium was recorded in West Bengal (Samanta et al., 2014).

Percentage positive samples of Paratyphoid Salmonella from broiler farms, layer farms and post-mortem cases with gastrointestinal lesions was recorded as 6.66%, 9.09% and 0.52% respectively.
 
Gross lesions in birds with Salmonellosis
 
The birds found positive for paratyphoid Salmonella infection were subjected to detailed necropsy examination. The birds were received with the history of anorexia, restlessness, dullness, depression and diarrhoea.

Carcasses were found to be septicemic (Fig 2A). Liver lesions comprised hepatomegaly, congestion with hemorrhagic and necrotic foci (Fig 2B) in liver. Splenomegaly along with congestion and mottling of spleen (Fig 2C) was observed. The caeca were inflamed and swollen. Severe haemorrhagic gastroenteritis and haemorrhagic typhlitis along with haemorrhagic caecal tonsils was observed. Presence of necrotic debris (caecal cores) in both the caeca (Fig 2D) was also an important finding. The lesions in ovary included in Batch-3 comprised of numerous ovarian follicles having congestion. The layers found to be Salmonella positive in our study were of higher age group and coagulated yolk sacs and stalk formation was not noted.

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The two most consistently observed features of paratyphoid infections in mature poultry are intestinal colonization and systemic dissemination to internal organs (Gast, 2013).
 
Histopathological lesions in birds with Salmonellosis
 
Liver
 
Microscopic lesions in liver included congestion, haemorrhages, hemosiderosis, congestion, dilatation of sinusoids, vacuolar degeneration, coagulative necrosis and cellular infiltration (Fig 3A). Presence of multifocal necrosis is an irreversible pathologic alteration. Kupffer cell hypertrophy was also noted. Liver was noted with maximum histopathological alterations in our study.

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Spleen
 
Spleen showed lymphocytic follicle depletion, micro haemorrhages and fibrinoid necrosis (Fig 3B). Similar findings reported in previous studies (Islam et al., 2006; Kumari et al., 2013).
 
Intestine
 
Intestine showed haemorrhages, desquamation of the epithelium and goblet cell hyperplasia. Intense cellular infiltration in the caeca and intestine was observed (Fig 3C). The findings were comparable to the microscopic lesions observed (Muna et al., 2016). Haemorrhages with infiltration of mononuclear cells in the intestinal submucosa were also observed in our study similar to the previous findings (Dutta et al., 2015). Special staining was done using Masson Trichrome staining in liver (Fig 3E) and intestinal sections (Fig 3F) and presence of increased amount of connective tissue was noted.
 
Ovary
 
Histopathological findings were noted in ovaries of layers included haemorrhages and huge cellular infiltration (Fig 3D).
 
Biofilm assay
 
Formation of blue coloured biofilm stained by crystal violet at the bottom of wells and on walls of the wells at air-liquid interface/pellicle biofilm was noted on visual observation. From our results, the cut-off value (ODC) 0.174 at OD of 630 nm was used to categorize test isolates.

The result showed that 40.0% isolates possessed biofilm producing ability where 20.0% isolates were weak biofilm producers and were obtained from the necropsy cases while 20.0% isolates were moderate biofilm producers belonging to layer farms. Also, the OD value for motile salmonellae was higher than that of non-motile salmonellae in our study while 60.0% isolates were non-biofilm producers.
 
Antibiotic sensitivity test
 
Based on the antibiotic sensitivity pattern, we could elucidate that multi-drug resistance (MDR) pattern was observed in the bacteria. Out of the isolated organisms, motile Salmonella isolates were resistant against oxytetracycline (100%), streptomycin (66.6%) and amoxicillin (33.3%) with 66.6% non-typhoidal Salmonella isolates resistant to two or more than two antibiotics. Multi-drug resistance pattern was observed in 37.5% non-motile Salmonella isolates where maximum resistance was observed against oxytetracycline (62.5%), streptomycin (25%) followed by resistance towards enrofloxacin and amikacin (12.5% each).

Since sampling was done in commercialized broiler and layer farms, we can observe that MDR pattern has become an important challenge for the management of poultry houses. The haphazard and irrational use of antibiotics has led to the resistance of Salmonella towards commonly used antibiotics which is likely to aggravate with passage of time.

Salmonellosis prevalence was found to be 20.0% and 45.45% in broiler and layer farms, respectively, as established by microbial culture, with a positive rate of 1.58% in post-mortem instances. Using polyvalent antisera, the percentage of positive non-typhoidal Salmonella samples from broiler farms, layer farms and post-mortem patients with gastrointestinal lesions was 6.66%, 9.09% and 0.52%, respectively. With the use of PCR, 11.1% of the isolates tested positive for Salmonella enterica serovar Enteritidis, a motile paratyphoid bacterium. S. Enteritidis was found to be present in 3.84% of the farms that were tested. The bacteria were discovered to be multidrug resistant (MDR), with motile paratyphoid isolates showing higher resistance than non-motile isolates. As a result, ongoing disease surveillance, including monitoring of the organisms’ antibiotic resistance patterns, should be promoted.