DESCRIPTION OF PROBLEM
Our laboratory hypothesized that tracheal sampling may be a viable method for detecting Salmonella contamination in poultry. Very recent research from our laboratory suggested that tracheal inoculation is indeed possible, and that low doses of Salmonella administered directly into the trachea can cause systemic infection [1, 2]. Thus, if infection does occur through respiratory inoculation, Salmonella should be able to be isolated from the trachea and may be a reliable organ for detection purposes. The purpose of this study was to compare the frequency of isolation of Salmonella between tracheal, cecal, and liver and spleen samples under a variety of environmental and commercial conditions in several countries in North and South America. Environmental humidity and temperature were measured to evaluate their effects on frequency of isolation from the organs.
MATERIALS AND METHODS
Culture Methodologies
Conventional methodologies were used for the isolation and enrichment of Salmonellae in the laboratories from each of these trials. Trachea, ceca, and liver and spleen samples were aseptically collected for determination of Salmonella incidence. In trials 2, 3, and 5, samples were enriched with tetrathionate enrichment broth [3]. Samples were pre-enriched in peptone water [4] for 8 h and then enriched with doublestrength tetrathionate enrichment broth in trials 1 and 4. Detection agars were McConkey agar [5] in trial 1, brilliant green agar [6] with 25 µg/ mL of novobiocin [7] in trial 2, xylose lysine deoxycholate agar [8] with novobiocin in trials 3 and 4, or xylose lysine tergitol-4 agar [9] in trial 5. The presence or absence of typical lactosenegative colonies of Salmonella was determined after enrichment incubation at 37°C for all trials, followed by selective plating on agar. In trials 1, 3 and 5, Salmonella serogroup was confirmed with poly-O Salmonella-specific antiserum [10].
Field Trial 1
A diagnostic laboratory for a poultry company in Bucaramanga, Santander, Colombia, participated in this study. Tracheas were sampled from broiler chickens of various ages over a period of 16 mo, ranging from January 2012 to April 2013, involving conventional commercial broiler farms associated with the company. A total of 1,061 samples were collected over this period of 13 mo, and the previously mentioned culture methodologies were employed for Salmonella isolation. Average maximum and minimum temperature for the area was recorded to be in the range of 25.3 to 18.9°C and the average air humidity was recorded to be 82% for the monitored period.
Field Trial 2
A commercial poultry company from Buenos Aires, Argentina, participated in this study during December 2012, and involved 4 conventional commercial broiler farms housing 80,000 broiler chickens in each farm. Average maximum and minimum temperature for the area was recorded to be in the range of 21 to 28°C and the average air humidity was recorded to be 63% for the monitored period. Chickens were screened via meconium sampling in the hatchery for Salmonella and were deemed negative. On d 3 and 13, all farms were treated with a proprietary water applied nutritional supplement with potential to reduce Salmonella, increase flock health, and increase production parameters. At 28 d of age, 5 chicks from each farm were cultured for Salmonella recovery in trachea, ceca, and liver and spleen.
Field Trial 3
A commercial broiler farm from Guanajuato, Mexico, participated in this trial, which was performed in the month of July 2012. Average maximum and minimum temperatures for the area were recorded at 18 and 28°C and the average air humidity was recorded at 65%. One hundred 49-d-old broiler chicks from the farm under study were cultured for enumeration of Salmonella incidence in ceca and trachea. Standard drag swab technique was employed for the house, with subsequent enrichment and selective plating as previously described. Drag swabs were assembled as previously described by Caldwell et al. [11]. Standard sterile cotton swabs were used for taking drag swabs of each fan duct in the house, with subsequent enrichment and selective plating as described above.
Field Trial 4
A commercial turkey farm from Arkansas was sampled in December 2011. Average minimum and maximum temperature for the area was recorded to be in the range of −2.9 and 4.9°C and the average air humidity was recorded at 67%. One hundred 16-wk-old female turkeys were cultured for Salmonella incidence in trachea and ceca.
Field Trial 5
A commercial broiler company from Arkansas participated in this study. The trial was performed during the months of January and February 2013 and involved 6 conventional commercial broiler farms. Average minimum and maximum temperatures for the area were recorded at −2.7 and 7.7°C and the average air humidity was recorded at 69%. Chickens were screened in the hatchery for Salmonella and were deemed negative. At approximately 24 d of age, 25 chicks from each farm were cultured for Salmonella recovery in trachea, ceca, and liver and spleen.
RESULTS AND DISCUSSION
Aerosolization is a traumatic process for most microorganisms, and survival can be dependent on the mechanisms of aerosolization, the climate into which these organisms are launched, the distance they are traveling, and time involved in the whole process. Salmonella has proven to be viable in laboratory-generated aerosols for more than 2 h [12]. Likewise, it has been shown that the death rate of Salmonella was influenced by the protective nature of the media during aerosolization, along with overall prevailing RH and temperature of the air [13]. Environmental temperature, humidity, and temperatures within litter may have a role in supporting the continued survival of the organism in dust and aerosol generated in the poultry facility. In fact, humidity is known to play a major role in survivability of Salmonella in aerosols, and dependence on humidity is known to be the characteristic of many gram-negative organisms [14]. The average minimum and maximum temperatures and average humidity were recorded at the locations in which the field studies herein were conducted.
In the present study, 5 field trails were conducted to determine the recovery rate of Salmonella from the trachea of commercial poultry. Ceca and liver and spleen samples were also collected for comparison to traditionally accepted sampling methods of detection (except for field trial 1); temperature and humidity measurements were recorded to determine the effects these parameters may have on the ability to detect Salmonella contamination in poultry under commercial conditions. All data for this report are represented in Table 1. In trial 1, conducted with a diagnostic laboratory of a poultry company from Bucaramanga, Colombia, a total of 1,061 cases of broiler chickens were tested for presence of Salmonella, of which 96 (9.04%) enriched tracheal samples were found to be positive. Serogroup determination showed that 89 (92.7%) of the samples belonged to group B, confirmed to be Salmonella Heidelberg, and 7 (7.3%) belonged to group A (data not shown). Trials 2 and 3 evaluated the presence of Salmonella in environmental samples, with enumeration of colony-forming units per gram of litter (Trial 2) and incidence of Salmonella in litter through drag swabs (Trial 3), and approximately 104 cfu of Salmonella was estimated to be present per gram of litter in trial 2, along with 7 of 8 (87.5%) litter drag swabs and 5 of 10 (50%) fan duct swab samples being positive for Salmonella in trail 3 (data not shown). These results correlated with the incidence of recovery of Salmonella from the trachea of birds at 8 of 20 (40%) in trial 2 and 28 of 100 (28%) in trial 3. These data support previously described studies demonstrating the presence of Salmonella in aerosols and dust inhaled by poultry in grow-out barns [11, 13, 15–19].
Despite a low sample number in trial 2, a relatively high incidence of tracheal samples (8/20; 40%) were positive for Salmonella, which was similar to levels reported in trials 3 and 4 [28/100 (28%) and 34/100 (34%), respectively]. Tracheal recovery was low in trial 5 (3/150; 2%) despite ceca and liver and spleen incidence similar to other trials, suggesting that even low levels of tracheal contamination can be an indicator of infection, with respect to our hypothesis; however, this might not be the sole explanation. Additionally, despite previous reports of an association between high environmental temperature and humidity and increased Salmonella incidence [13, 20–22], such a correlation was not noted in the present study. The highest temperatures and humidity, 18.9 to 25.3°C and 82%, were recorded in trial 1, which had only 9.04% tracheal recovery of Salmonella. However trials 2, 3, and 4 each reported similar or relatively higher levels of Salmonella recovery, despite wide variations in temperature and humidity. Therefore, a previously reported association between high environmental temperature and humidity and increased Salmonella incidence was nonexistent, as far as the present study is concerned. In summary, Salmonella was recovered from tracheal samples in all trials evaluated in the present study. These data support the hypothesis that tracheal samples can be an indicator of Salmonella contamination, and such contamination is likely an indication of infection, as evidenced by positive recovery in gastrointestinal and liver and spleen sampling
Table 1. Evaluation of recovery of Salmonella in trachea, ceca, and liver and spleen in commercial chickens and turkeys
CONCLUSIONS AND APPLICATIONS
- These data offer further evidence that airborne transmission may be important for Salmonella transmission within and between poultry flocks.
- The results of these studies suggest tracheal recovery of Salmonella is a viable and sensitive site for detection of Salmonella infection.
- Tracheal samples can be used as a complementary tissue to ceca and liver and spleen to increase Salmonella recovery in positive poultry.
- Tracheal Salmonella recovery was effective even with the variations in temperature and humidity in the trials.
REFERENCES AND NOTES
- Tellez, G. 2013. Technologies for Salmonella control in live production. The Poultry Federation. Accessed July 2013. http://www.thepoultryfederation.com/public/userfiles/files/Tellez%20Salmonella%20Summit%202013.pdf.
- Tellez, G., J. Latorrez, L. Bielke, A. Menconi, O. Faulkener, A. Wolfenden, and B. Hargis. 2013. Detection of Salmonella from trachea in commercial poultry as an epidemiological tool. International Poultry Scientific Forum 2013. Accessed July 2013. http://www.ippexpo.org/ipsf/ docs/13AbstractBook.pdf.
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