A case control study of Salmonella SPP. infection in stray dogs in Tehran shelters and the correlation between paraclinical tests results and clinical findings

Introduction

Zoonotic diseases caused by Salmonella spp. are widespread and observed in most animals. The incidence rate of such diseases has recently increased in humans and animals, especially dogs, as compared to last four decades ( Zahraei Salehi, 1999 ; Schmidt and Tirado, 2001 ; Tsai et al., 2007 ). Salmonella is the etiologic agent of salmonellosis which is a very common intestinal infection in humans and animals. The bacteria are among the most common causes of acute and chronic diarrhea, as well as mortality in human and different livestock ( Thompson et al., 2001 ).

The prevalence of Salmonella spp. in dogs is not fully understood since dogs can serve as asymptomatic carriers and excrete the bacteria with no clinical demonstrations. The dogs carrying Salmonella spp. can excrete the bacteria for more than 6 weeks ( Greene, 1998 ; Marks and Kather, 2003 ). The clinical symptoms of salmonellosis in dogs include fever, loss of appetite, diarrhea, bloody diarrhea, abdominal pain, and abortion ( Greene, 1998 ; Marks and Kather, 2003 ). A part of these symptoms usually occurs 3-5 days after infection with the bacteria; nonetheless, the clinical symptoms may appear even 12 hours after exposure to bacteria ( Marks et al., 2011 ). Nevertheless, the clinical salmonellosis infection in dogs is rare and the subclinical form of the disease is the dominant type in dogs ( Sanchez et al., 2002 ). Dogs kept in animal shelters can be a good source for the distribution of different serovars of Salmonella in the environment ( Jackson et al., 2013 ). Animals, especially carnivores, as the asymptomatic carriers of Salmonella spp. play a significant role in this cycle. However, most of the studies in Iran and many other countries have focused on salmonellosis in poultry populations. Different reports have highlighted the transmission of antibiotic-resistant Salmonella spp. from animals to humans living in contact ( Sanchez et al., 2002 ). Although Salmonella serovars are severely host-limited, they can contaminate a wide range of hosts ( Ojo and Adetosoye, 2009 ). Therefore, determining the antibiotic susceptibility pattern of Salmonella species isolated from such animals can provide valuable information for the identification of ineffective medicines and replacing them with useful ones in order to promote and maintain public health. With this background in mind, the current study aimed to evaluate the serologic and bacteriologic prevalence of salmonellosis among stray dogs placed in the animal shelters around Tehran, Iran, compare the results with those of asymptomatic dogs, and determine the serotypes, as well as antibiotic susceptibility patterns of the isolated species.

Material and Methods

A 10-mL blood sample was taken from each dog, transferred to blood culture bottle, and subcultured on MacConkey and Salmonella-Shigella agar plates after 24, 48 and 72 h. The current study was conducted on 100 fecal swab samples obtained from dogs with clinical symptoms of diarrhea, loss of appetite, and fever (as the case group) and another 100 fecal swab samples collected from apparently (clinically) healthy dogs or those recovered from the abovementioned symptoms (as the control group) placed in four animal shelters around Tehran from spring 2016 to winter 2017. The fecal specimens were collected in the feces sampling containers. The rectal swab technique was also employed if the fecal specimen was not available.

Culture test. The specimens were transferred on ice to laboratory, and the samples were cultured on selective medium on the day of sampling. For this purpose, the specimens were initially cultured directly in Rappaport Vassiliadis enrichment broth and were inoculated on the selective media as MacConkey and Salmonella-Shigella agar plates after 24 hours. After performing the biochemical differentiation tests, three colonies from each medium were stored at -20°C. Moreover, to determine the genotype of the microbiologically identified species, DNAs of the stored species were extracted. Thereafter, the polymerase chain reaction (PCR) was used to confirm the identifications with four pairs of primers ( Fratamico, 2003 ). In addition, the food of the dogs was cultured every 20 days in order to monitor contamination with Salmonella spp. To trace bacteremia, blood samples taken from the animals were cultured. For this purpose, after shaving, the area was cleaned with alcohol-soaked cotton balls and the blood samples were then taken based on the standard guidelines ( Quinn et al., 1994 ). A 1mL blood sample was obtained from each dog, cultured on MacConkey and Salmonella-Shigella agar plates, and biochemically assessed based on the fecal culturing technique in order to evaluate bacteremia.

Serological evaluation and serotyping. The tube agglutination test was performed using the Widal antigens (Iran Pasteur Institute, Karaj, Iran) ( Waltman et al., 1998 ), based on manufacturers instruction. Serotyping was performed to confirm the results of microbiological tests, and serum grouping was conducted based on manufacturer’s instructions (Difco Co., Maryland, USA) and specific antisera ( Agasan et al., 2002 ).

Antibiotic susceptibility pattern. After the isolation and identification of Salmonella species using the Clinical and Laboratory Standards Institute (CLSI) guidelines and standards, the Kirby-Bauer disk diffusion method was used to determine the susceptibility pattern of the species. To this end, after providing a bacterial suspension adjusted to 0.5 McFarland turbidity standard, the sterile swabs were used to inoculate the suspension onto Mueller-Hinton agar plates. Subsequently, four disks of gentamicin, amikacin, streptomycin, and ciprofloxacin (Mast Co., Merseyside, United Kingdom) were planted on the plates ( Salehi et al., 2005 ).

Statistical analysis. The obtained results were analyzed and compared using Chi-squared test at the probability level of %5.

Results

In general, as evidenced by the results of the current study, a total number of 100 samples were obtained from clinically symptomatic dogs. No significant difference was detected between the case and control groups in terms of gender (60 female and 61 male dogs) (P=0.89). In addition, there were no significant differences between the groups regarding length of shelter stay (P=0.81). Some symptoms, including fever, lethargy, diarrhea, and abdominal pain, were observed in all symptomatic dogs (case group). Moreover, the most and least common clinical symptoms were reported as loss of appetite and bloody diarrhea with 90% and 22% prevalence. In addition, among the evaluated symptoms, bloody diarrhea and leanness were the most commonly observed symptoms in the Salmonella-positive cases, compared to the negative subjects. Moreover, a significant correlation was observed between tube agglutination positivity and bloody diarrhea/ leanness in the evaluated dogs.Based on the culture results of 100 fecal swab samples obtained from the case group with suspected salmonellosis, 11 samples (11%) were positive for Salmonella spp. The results of the repeated cultures were the same as what was obtained in the first one. Similar results were also reported from the same number of fecal swabs (n=100) obtained from the control group dogs with 4 positive cases (4%). In addition, the results of food culturing were negative, similar to those of blood culture test. The tube agglutination test was positive for 12(12%) and 5 (5%) serum samples in the case and control groups, respectively. The number of positive samples obtained from culture and tube agglutination tests in the case group was significantly higher, as compared to that of the control group (P=0.03 and P=0.003, respectively). Out of 11 samples with positive culture, 7 cases were identified as S. typhimurium and 4 as S. enteritidis. No significant differences were observed between the groups in terms of the type of serovars (P=0.49). Polymerase chain reaction (PCR) was amplified on the positive samples of both control and case groups grew on selective media and the results of PCR were the same as those achieved in the culture test (Figure 1). The antibiotic susceptibility testing was performed on the species grown on the selective media, the results are displayed in Table 1. The highest resistance was observed in S. enteritidis against gentamicin, as well as S. typhimurium against amikacin (Table 1). No significant difference was reported between the serovars in terms of antibiotic resistance (P>0.05).

Figure 1. Gel electrophoresis image of polymerase chain reaction products.

Discussion

While animals do not contribute to the epidemiology of typhoid fever, they play a significant role in the epidemiology of salmonellosis caused by the other serotypes of Salmonella ( Zahraei Salehi, 1999 ). There exists a massive population of stray dogs in cities with no certain monitoring and control systems over their nutritional habits. Consequently, these animals can pose a serious challenge to public management in urban communities since they can play a significant role in the transmission of zoonotic serovars to humans ( Kaufmann, 1966 ; Sato et al., 2000 ). As a matter of fact, stray dogs are considered the major causes of global failure of programs which were developed to control or eradicate Salmonella in animal ( Sato et al., 2000 ). Therefore, the provision of information regarding salmonellosis in dogs, particularly the carrier status, can be of great help in promoting public health.

Since salmonellosis in dogs is usually subclinical and asymptomatic, the mysterious role of dogs in the transmission of the infection should be investigated in further studies ( Zahraei Salehi, 1999 ). It is rather difficult to diagnose salmonellosis based on clinical symptoms since none of the symptoms is disease-specific and most of them are common among different diseases. Therefore, it is necessary to consider symptoms of salmonellosis along with laboratory results ( Zahraei Salehi, 1999 ). The traditional conventional diagnosis of salmonellosis in dogs relies on the isolation of microorganisms corresponded to the targeted clinical symptoms, as well as the evaluation of risk factors, such as hospital stay, age, environmental factors, and antibiotic administration ( Jajere et al., 2014 ). Several culture tests should be performed in the case of diagnosis merely based on microbiological findings, ( Marks and Kather, 2003 ) since the sensitivity and specificity of culture test is significantly lower than PCR ( Schuurman et al., 2007 ). The isolation of Salmonella spp. was also reported from apparently healthy dogs ( Kaufmann, 1966 ; Tanaka et al., 1976 ; Sugiyama et al., 1993 ; Weber et al., 1995 ). In general, the prevalence of Salmonella species in asymptomatic dogs is estimated at 0-43% ( Jajere et al., 2014 ). In their study, Ojo and Adetosoye (2009) failed to detect Salmonella spp.in the intestinal contents of 100 stray dogs. However, Sanchez et al. (2002) pointed to the intestinal carriage of Salmonella spp. by 36% of healthy dogs. The results of the current study were in agreement with those obtained by Seepersadsingh et al. (2004) who reported 3.6% prevalence for the non-diarrheal Salmonella-carrier dogs. On the other hand, the results of the present study were inconsistent with a study that reported the prevalence of 20% for the Salmonella- carrier dogs ( Jajere et al., 2014 ). Nonetheless, the results of the later study was in agreement with those of aforementioned studies since the non-diarrheal asymptomatic stray dogs can be the carriers for the bacteria and play the role of reservoir by excreting the bacteria to the environment. The isolation of Salmonella species from the fecal specimens of healthy dogs may lead to misleading interpretations, particularly in the case of diarrheal animals ( Cantor et al., 1997 ). However, the clinical salmonellosis has also been reported in dogs, and in case of the exacerbation of this disease, nausea, vomiting, fever, depression, abortion, and even death may occur ( Jajere et al., 2014 ). The prevalence of Salmonella spp. among the stray dogs dropped from 23.5% in 1970 to 5.5% in 1975 ( Jajere et al., 2014 ). Hackett and Lappin (2003) performed a study on the prevalence of intestinal diseases among dogs based on the findings from fecal specimens. They indicated that Salmonella species account for 3.2% of factors which cause diarrhea in these animals. Based on the results of a study carried out by Tsai et al. (2007) , the prevalence of Salmonella spp. among pet and stray dogs was 2.1% and 6.3%, respectively with Salmonella enterica var. Düsseldorf dominance in the both groups. Hackett and Lappin reported that the prevalence of Salmonella spp. among the stray dogs was 12% which was higher than the rate reported in the studies conducted by Tsai et al. (2007), lower than that obtained by Khan ( Hackett and Lappin, 2003 ). Nonetheless, it was very close to the rate indicated by Salehi et al. (2013) who reported that about 10.5% of stray dogs living around Garmsar, Iran, carried Salmonella species. Based on the annual report of the United States Department of Agriculture (USDA), the National Veterinary Services Laboratories (NVSL) 2011, S. typhimurium and S. enterica serotype Senftenberg, followed by S. enterica serovar Muenchen, Newport, and Javiana were the most common Salmonella species and serotypes in dogs ( Jajere et al., 2014 ). However, the dogs even can carry a wider range of serovars. Morse and Duncan isolated 35 different Salmonella serovars from dogs, while Seepersadsingh isolated 28 different serovars. Nevertheless, S. typhimurium was the most common species isolated from these animals ( Seepersadsingh et al., 2004 ; Jajere et al., 2014 ). The antibiotic susceptibility pattern of the species isolated in the current study was rather alarming. Therefore, it can be concluded that indiscriminate use of antibiotics in animals may result in the emergence of new antibiotic-resistant species. Consequently, the management of antibiotics should be monitored properly and only be prescribed after the confirmation of their efficacy ( Ojo and Adetosoye, 2009 ). In the current study, the lowest resistance was against ciprofloxacin which was in line with the results of the study conducted by Tsai et al. (2007). The reported susceptibility to ciprofloxacin and ceftriaxone in all the isolated Salmonella species. On the other hand, they indicated the highest resistance against tetracycline (77.5%), followed by chloramphenicol (52.5%) and ampicillin (50%) ( Tsai et al., 2007 ). Zoonotic diseases are regarded as public health concerns, and dogs are the active carriers of Salmonella species which is one of the most important human pathogens. In light of the abovementioned facts, the present study pointed to the great importance of regular monitoring of stray dogs, particularly the ones placed in shelters. It is due to the fact that high microbial continuous contamination in such places is a potential risk for public health and can cause outbreaks in dogs and consequently in humans, if being neglected.

Ethics

We hereby declare all ethical standards have been respected in preparation of the submitted article.

Authors’ Contribution

Study concept and design: Siamak Mashhady Rafiee

Acquisition of data:Nader Askari

Analysis and interpretation of data: Nader Askari

Drafting of the manuscript: Nader Askari

Critical revision of the manuscript for important intellectual content: Siamak Mashhady Rafiee

Statistical analysis: Kumarss Amini

Administrative, technical, and material support: Siamak Mashhady Rafiee

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