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Arquivo Brasileiro de Medicina Veterinária e Zootecnia

Print version ISSN 0102-0935

Arq. Bras. Med. Vet. Zootec. vol.66 no.5 Belo Horizonte Oct. 2014

http://dx.doi.org/10.1590/1678-6716 

Veterinary Medicine

Isolation of Salmonella enterica subsp. enterica (O:4,5:i) and Salmonella enterica subsp. Typhimurium from free-living domestic pigeons (Columba livia)

Detecção de Salmonella enterica subsp enterica (O:4,5:i) e Salmonella enterica subsp. Typhimurium em pombo doméstico de vida livre (Columba livia)

R.C. Rocha-e-Silva 1  

W.M. Cardoso 1  

R.S.C. Teixeira 1  

Á.H. Albuquerque 1  

R.V. Horn 1  

E.S. Lopes 1  

V.J.R. Gomes Filho 1  

C.P. Almeida 1  

I.C.L. Santos 1  

D.N. Machado 1  

S.V.G. Lima 1  

I.S. Carneiro 2  

1Universidade Estadual do Ceará - UECE - Fortaleza, CE

2Universidade de Fortaleza - Unifor - Fortaleza, CE

ABSTRACT

The present study reports the isolation of Salmonella enterica in organs of free-living domestic pigeons. In the clinic examination, the presence of feces in the peri-cloacal and abdominal regions were observed, as well as symptoms such as cachexy, incoordination and opisthotonos. Before any therapeutic protocol was applied the bird died and a necropsy was then performed for the removal of spleen, liver, kidney and intestine for bacteriological examination and antibiotic sensitivity test. Salmonella enterica subsp.enterica (O:4,5:i-) and Salmonella enterica subsp. enterica serovar Typhimurium were isolated from the liver and intestine and the sensitivity test demonstrated that these strains are sensitive to several antibiotics.

Key words: isolation; necropsy; bacteriological examination; sensitivity

RESUMO

O presente trabalho relata o isolamento de Salmonella enterica em órgãos de um pombo doméstico de vida livre. No exame clínico foi observada a presença de fezes pericloacal na região ventral, caquexia, incoordenação motora e opistótono. Antes de iniciar um protocolo terapêutico, a ave foi a óbito, e, em seguida, foi realizada uma necropsia para remoção do baço, fígado, rim e intestino para exame bacteriológico e teste de sensibilidade a antibióticos. Foi isolado Salmonella enterica subsp. enterica (O:4,5:i-) e Salmonella enterica subsp. enterica sorovar Typhimurium do fígado e intestino, e o teste de sensibilidade demonstrou que essas cepas são sensíveis a vários antimicrobianos.

Palavras-Chave: isolamento; necropsia; exame bacteriológico; sensibilidade

INTRODUCTION

The pigeons are reservoirs for several organisms that are pathogenic for animals and humans. Approximately 60 different microorganisms, including viruses, bacteria, fungi and protozoan were isolated from pigeons (Haag-Wackernagel e Moch, 2004).

Among the pathogens found in these birds is Salmonella enterica, a bacteria responsible for salmonellosis, which is the most importance zoonotic disease in public health, responsible for outbreaks of gastrointestinal illness in humans (Hauri et al., 2004) and was isolated from feces of domestic pigeons (González-Acuña et al., 2007).

The molecular epidemiological studies also indicate that different clones of serovar Typhimurium predominate among passerines, gulls, and pigeons (Refsum et al., 2002) and the serotype Typhimurium is frequently associated with disease in many different mammalian and avian host species (Rabsch et al., 2002). However, S. Typhimurium is not adapted to any particular host animal (Grund and Stolpe, 1992).

Due to the survival of salmonelas in a natural environment, salmonellosis continues to be an important zoonosis, and can be transmitted to humans through animal and contaminated food. The pigeon is an important source of infection (Grund and Stolpe, 1992).

Thus, the aim of the present study was to report the occurrence of Salmonella spp. in free-living domestic pigeons and the antimicrobial resistance profile of these micro-organisms.

CASUISTRY

An adult male free-living domestic pigeon (Columba livia) was brought to the Laboratory of Ornithological Studies (LABEO) of State University of Ceará (UECE), Brazil. Upon the physical exam the bird presented cachexy, incoordination, opisthotonos and the presence of feces in the pericloacal and ventral regions. Before any therapeutic protocol was applied, the bird died and a necropsy was performed aseptically for removal of spleen, liver, kidney and intestine for bacteriological examination.

The organs were macerated individually in a petri dish and transferred to tubes containing Buffered Peptone Water (DIFCO, Sparks, Maryland, USA) and incubated for 24h at 40°C. After incubation, the aliquots were transferred to tubes containing Selenite-Cystine broth (DIFCO, Sparks, Maryland, USA) and Rappaport-Vassiliadis broth (DIFCO, Sparks, Maryland, USA). Subsequently, the samples were plated onto Brilliant Green agar (DIFCO, Sparks, Maryland, USA) containing 40µg Novobiocin (SIGMA), Salmonella-Shigella agar and MacConkey agar (DIFCO, Sparks, Maryland, USA). Colonies suspected to contain Salmonella were collected from each plate for biochemical identification using TSI (Triple Sugar Iron Agar), LIA (Lisine Iron Agar), SIM (Sulfur, Indol, Motility), VP (Voges-Proskauer), MR (Methyl Red), Urea, Ornithine Descarboxylase Broth and Simmons Citrate Agar. The temperature and period of incubation were standardized at 40°C for 24h.

Colonies with biochemical profile of Salmonella were submitted to slide agglutination test using a polyvalent serum against O antigen (Probac). The colonies that agglutinated during one or two minutes were considered positive for Salmonella, and were preserved in Nutrient Agar. Isolates were submitted to FioCruz laboratory in Rio de Janeiro, Brazil, for complete identification and serotyping.

In order to determine the antimicrobial resistance profile, the disk diffusion method on Muller-Hinton Agar was tested according to CLSI (2003). The following antimicrobial agents were tested: penicilin G (10U), tetracycline (30µg), gentamicin (10µg), chloramphenicol (30µg), ciprofloxacin (30µg), ampicillin (10µg), streptomycin (10µg), sulfonamide (300mcg), neomycin (30µg), thiamphenicol (30µg) and erythromycin + sulfonamide + trimethoprim (15mcg + 300mcg + 5mcg). Following the application of the antimicrobial disks, the inoculated plates were incubated at 37°C for 24h. The diameters of the inhibition zones were measured (millimeter) and were compared to internationally accepted measurements to determine the susceptibility or resistance of the isolate. Salmonella Pullorum ATCC strain was used as control.

Salmonella enterica subsp. enterica (O:4,5:i) (S. enterica O:4,5:i) was isolated from the liver sample and Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) was isolated from liver and intestine samples. The results from the antimicrobial resistance test of the Salmonella isolated from samples of pigeon organs are shown in Tab. 1. S. enterica O:4,5:iwas resistant to streptomycin, penicillin, thiamphenicol and neomycin, while S. Typhimurium was resistant to streptomycin, penicillin and thiamphenicol.

Tabela 1 Antibiotic resistance profiles of Salmonella enterica subsp.enterica (O:4,5:i-) and Salmonella enterica subsp. enterica serovar Typhimurium isolated from organs of a naturally infected pigeon 

Antimicrobial agent
Isolate
AM CP CHL GM TE ST SF P TF NM EST
Salmonella enterica subsp.enterica (O:4,5:i)
S S S S S R S R R R S
SalmonellaTyphimurium
S S S S S R S R R S S

AM: ampicillin (10µg); CP: ciprofloxacin (30µg); C: chloramphenicol (30µg); GM: gentamycin (10µg); TE: tetracycline (30µg); ST: streptomycin (10µg); SF: sulfonamide (300mcg); P: penicillin (10U); TF: thiamphenicol (30µg); NM: neomycin (30µg); EST: estreptomicin+sulfonamide+trimethoprim (15mcg+300mcg+5mcg); S: susceptible; R: resistant.

DISCUSSION

The pigeons have been appointed as transmitters of pathogenic microorganisms to humans (Haag-Wackernagel and Moch, 2004), birds and other animals (Jahantigh and Nili, 2010).

Several studies performed with pigeons report the presence of pathogenic agents, mainly the ones that compromise the public health and cause significant economic impact worldwide, such as Salmonella spp. (Trevejo et al., 2003).

In this study, two distinc serovars were isolated, S. enterica O:4,5:i and S. Typhimurium from two organs: liver and intestine. Both were isolated from the liver, while only S. Typhimurium was isolated from the intestine.

According to Grund and Stolpe (1992), the best site to isolate the pathogen is in the incision of the bowels, mainly in the ileum, although the small and large intestines may present the bacteria as well, but in smaller number.

The intestine samples that were positive for Salmonella were removed from two distinct portions: cranial (small intestine) and caudal (large intestine). The pathogen was successfully isolated from both portions.

The other isolate, S. enterica O:4,5:i, member of the O:4 group, the same as the S. Typhimurium, was not, to the best of the author's knowledge, reported causing infection in pigeons or any other animal species, neither in environmental, economic nor in public health nuisance.

The antimicrobial sensitivity test performed demonstrated that both S. Typhimurium as well as S. enterica O:4,5:I presented sensitivity to almost all of the antimicrobial agents.

The resistance of S. Typhimurium to streptomicin and sulfonamide has been widely reported in studies performed with isolates from feces of humans with gastroenteritis, contaminated feed and animal sources (Gorman and Adley, 2005) and the high resistance rate to multiple drugs are constantly present in many serotypes that cause human salmonellosis (Lauderdale et al., 2006).

The resistant microorganisms may be transferred to humans through contact with sick animals or consumption of contaminated food originated from animals. Therefore the antimicrobial resistance monitoring in animals is highly important due to the risk of resistance gene transmission between pathogenic bacteria and the transmission of these pathogens to humans through the consumption of products derived from these animals (Yang et al., 2001). Therefore, the resistance profiles are the result of the use of antimicrobial agents in human medicine, as well as in the animal originated food industry (Lauderdale et al., 2006).

According to the genotype and the profile of antimicrobial resistance, the strains of Salmonella associated with captive and free-living birds represent a potential threat to men, since they can act as reservoirs for the transmission of salmonelas to the pet owners and people that live in direct or indirect contact with such birds (Hudson et al., 2000).

CONCLUSION

In conclusion, the free-living pigeons are capable of hosting Salmonella enterica subsp.enterica (O:4,5:i-) and Salmonella enterica subsp. enterica serovar Typhimurium, which may be zoonotic and are resistant to drugs commonly used to treat human infections.

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Received: April 02, 2013; Accepted: February 19, 2014

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