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10-1995

Identification of Salmonella enteritidis fromExperimentally Infected Hens Using a ColorimetricDNA Hybridization MethodP F. CotterFramingham State College

J E. MurphyGENE-TRAK Systems, Inc.

J D. KlingerGENE-TRAK Systems, Inc.

Robert L. Taylor Jr.University of New Hampshire

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Recommended CitationCotter, P. F., Murphy, J. E., Klinger, J. D. and Taylor, R. L. Jr. Identification of Salmonella enteritidis from Experimentally Infected HensUsing a Colorimetric DNA Hybridization Method. Avian Diseases. Vol. 39, No. 4 (Oct. - Dec., 1995) , pp. 873-878.

Identification of Salmonella enteritidis from Experimentally Infected HensUsing a Colorimetric DNA Hybridization Method

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Identification of Salmonella enteritidis from Experimentally Infected Hens Using a ColorimetricDNA Hybridization Method Author(s): P. F. Cotter, J. E. Murphy, J. D. Klinger, R. L. Taylor and Jr. Source: Avian Diseases, Vol. 39, No. 4 (Oct. - Dec., 1995), pp. 873-878Published by: American Association of Avian PathologistsStable URL: http://www.jstor.org/stable/1592426Accessed: 05-03-2015 19:56 UTC

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AVIAN DISEASES 39:873-878, 1995

Identification of Salmonella enteritidis from Experimentally Infected Hens Using a Colorimetric DNA Hybridization Method

P. F. Cotter,A J. E. Murphy,B J. D. Klinger,B and R. L. Taylor, Jr.c

ABiology Department, Framingham State College, Framingham, Massachusetts 01701 BGENE-TRAK Systems Inc., Framingham, Massachusetts 01701

CDepartment of Animal and Nutritional Sciences, University of New Hampshire, Durham, New Hampshire 03824

Received 3 May 1995

SUMMARY. Identification of Salmonella enteritidis from cloacally challenged commercial laying hens was studied by comparing bacterial isolations using conventional methods with detection by the use of the GENE-TRAK? colorimetric DNA probe assay. More positive test results were obtained using the latter on days 14, 28, and 42 postchallenge, but the difference between the two methods was not statistically significant. Over the duration of the experiment, positive cloacal samples were statistically more frequent from a commercial strain of white leghorn hens when compared with a commercial brown egg-producing strain (28/60 vs. 9/ 57; chi-square 1 df = 12.9, P < 0.001). Eggs having various shell defects were produced by the infected hens only after Salmonella challenge. These defects included, in order of fre- quency, elongated shape, thin shells, off-white color (tints), small size, wrinkles, and pimples. No Salmonella could be recovered from 193 defective eggs, nor were positive isolates made from additional tests performed on 50 normal eggs. Proteus sp. was isolated from 10 eggs, however. Our observations demonstrate that the GENE-TRAK colorimetric method is com- parable with conventional bacteriology for the identification of Salmonella in cloacal samples taken from laying hens. Moreover, the two methods demonstrate the existence of breed differences in susceptibility to S. enteritidis challenge.

RESUMEN. Identificaci6n de Salmonella enteritidis en gallinas infectadas experimental- mente mediante la utilizaci6n de un metodo colorim6trico de hibridaci6n del acido deso- xiribonucleico.

Se estudi6 la identificaci6n de Salmonella enteritidis a partir de gallinas ponedoras de- safiadas por la via cloacal. Se compararon los metodos convencionales de aislamiento bac- teriano con el uso de la sonda colorimetrica del acido desoxiribonucleico, Gene-Track?. Con este ultimo metodo se obtuvieron mayores resultados positivos a los 14, 28 y 42 dias despues del desafio, pero no hubo una diferencia estadisticamente significante entre los dos metodos. Durante el experimento, las muestras cloacales positivas fueron estadisticamente mas frecuentes en las gallinas de una estirpe comercial leghorn blanca que en una estirpe comercial de huevo marr6n (28/60 vs. 9/57: Chi-cuadrado 1 gl = 12.9 P < 0.001).

En las gallinas infectadas, se obtuvieron huevos con varios defectos en la cascara solamente despues del desafio. Estos defectos incluyeron huevos alargados, con cascara delgada, perdida de color, tamanio reducido y huevos con cascaras arrugadas y granulosas. No se pudo aislar la Salmonella de ninguno de los 193 huevos defectuosos ni de un grupo de 50 huevos normales. Solamente se aisl6 Proteus sp. de 10 huevos.

Los resultados demostraron que el m6todo colorimetrico Gene-Trak? es comparable con el metodo bacteriologico convencional para la identificaci6n de Salmonella en muestras tomadas de la cloaca de ponedoras comerciales. Ademas, los dos metodos demostraron di- ferencias en la susceptibilidad entre las estirpes frente al desafio con S. enteritidis.

Key words: Salmonella enteritidis, DNA hybridization, cloacal samples

This is Scientific Contribution No. 1828 of the New Hampshire Agricultural Experiment Station.

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P. F. Cotter et al.

Abbreviations: CB = conventional bacteriology; GT-DNAH = GENE-TRAK? DNA hybrid- ization; TSB = trypticase soy broth

Conventional methods for identification of Salmonella that employ selective and differ- ential culture media for their isolation, and car- bohydrate fermentation patterns for the speci- ation of suspect bacteria (13) have been sup- plemented by newer more rapid techniques. One such method is GENE-TRAK? DNA hy- bridization (GT-DNAH), in which the presence of Salmonella in a sample is determined by the

capture and detection of a species-specific rRNA target unique to Salmonella (4,5). The early version of this method that employed a short- lived radioactive reporter probe has been sup- planted by a second generation procedure that is colorimetric, which obviates some of the dif- ficulties associated with radioactivity (2).

The recovery of Salmonella from experimen- tally infected hens and their eggs was studied

by comparing the relative efficiency of the early, radioactive GT-DNAH method to one using conventional bacteriology (CB). In those stud- ies, more Salmonella were recovered using the GT-DNAH method than by CB, but the overall rate of infection was low (9,10). Additionally, some of the hens exposed to S. enteritidis via the cloacal route produced eggs with defective shells, 20% (10/49) of which also contained Salmonella (10).

The objectives of the present study were to compare CB with the newer colorimetric GT- DNAH by measuring the frequency of identifi- cation of S. enteritidis by both methods from cloacal swabs and eggs produced by hens after a cloacal challenge. Both a commercial white leghorn strain and a commercial brown egg- producing strain were included because sus- ceptibility differences between breeds have been reported with other salmonellae (12). The du- ration of such infections was also studied, as was the postchallenge production of eggs hav- ing various shell defects.

MATFRIATIS AND METHODS

Hens. Adult laying hens were housed in an iso- lation facility and were placed individually in wall- mounted, wire egg-collection cages having sloped bottoms. Twenty brown and 20 white leghorn com- mercial strain egg-production types were included in the study. They were fed a commercial formula layer

ration containing 20% crude protein and 2,860 kcal ME/kg. Feed and water were available ad libitum. All experimental hens were prechallenge negative for S. pullorum antibody, and negative for the presence of Salmonella bacteria as determined by sampling of their cloacas using both CB and GT-DNAH assay methods. Because of the frequency of isolating Pro- teus sp. from the eggs of hens challenged by S. en- teritidis in our earlier studies (9,10), half of the hens (10) of each strain were challenged with S. enteritidis alone or combined with a Proteus sp. It was our hope that such a combined challenge would result in a higher incidence of eggs positive for Salmonella.

Eggs from each hen were saved for approximately 14 days prior to the challenge. They were examined for the presence of the types of shell defects associ- ated with S. enteritidis as described in our earlier experiments (9,10). None were found.

Bacteria and challenge. A clinical isolate of S. enteritidis (CDC strain no. 1406-82) from human stool and a cloacal isolate of Proteus sp. obtained from a healthy hen were used as the challenge microorgan- isms. Log-phase trypticase soy broth (TSB) (BBL, Be- thesda, md.) cultures of each bacteria were diluted with additional TSB to the turbidity of a MacFarland No. 1 standard (Difco Laboratories, Detroit, Mich.), giving an estimated 3 x 108 bacteria per milliliter. One milliliter of S. enteritidis used alone, or com- bined with an additional 1 ml of the Proteus sp. cul- ture were deposited directly into the cloaca of each manually immobilized hen. The inocula were deliv- ered using a syringe fitted with a blunt catheter at a rate that allowed for the retention of nearly all of the liquid.

Sample collection. Cloacal samples were col- lected on postchallenge days 14, 28, and 42, using sterile cotton swabs that were placed immediately into tubes containing 10 ml of 1% peptone broth (Dif- co). Tissue sample sections were obtained aseptically at the termination of the experiment (day 50) from the ovary, the ceca, and the large bowel of each hen.

Eggs were collected daily, marked with the hen number, dated, and stored at room temperature until testing at the end of each of three collection intervals over the course of the experiment; on postchallenge days 14, 28, and 42. This practice was deemed nec- essary for pragmatic reasons so that sufficient numbers would be available to justify the expenditure of re- sources needed to conduct both CB and GT-DNAH. Groups of three unwashed eggs from individual hens were placed into a small plastic bag, broken by crush- ing, and their entire contents (shell included) were homogenized manually for 30 sec. One-milliliter ali- quots of such homogenates were used for further bac- teriological testing.

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S. enteritidis identification by DNA hybridization

Table 1. Identification of Salmonella from postchallenge cloacal swabs using CB and GT-DNAH.

~Testing ~Days postchallenge Testing Hen type method 14 28 42 Total

White CB 11/20^ 7/20 4/20 22/60B Brown CB 4/19 0/19 0/19 4/57 White GT-DNAH 14/20 9/20 5/20 28/60c Brown GT-DNAH 5/19 1/19 3/19 9/57

ANo. positive cloacal isolates/no. hens in group. BChi-square, total cloacal positives white vs. brown hens using CB = 9.6. P < 0.01. CChi-square, total cloacal positives white vs. brown hens using GT-DNAH = 6.8, P < 0.01.

Conventional bacteriology. Ten milliliters of 1% peptone broth cultures were incubated for 18 hr at 39 C, the pre-enrichment phase. One milliliter ali- quots including the swab (where applicable) were transferred to 10 ml tetrathionate broth (Difco) con- taining 0.01% brilliant green and sulfathiazole (Sigma Chemicals, St. Louis, Mc.) (1.25 Ag/ml) and incubat- ed for an additional 24 hr the selective enrichment phase. Brilliant green agar (Difco) and Hektoen en- teric agar (Difco) plates were streaked from the te- trathionate broths and these were incubated at 37 C for 24 hr. Suspect Salmonella colonies were trans- ferred into triple sugar iron agar (Difco) and lysine iron agar (Difco) slants and incubated overnight at 37 C. Slants showing reactions consistent with known positive controls were tested for carbohydrate fer- mentation reactions typical of Salmonella (3) using the Minitek? disk system (BBL). Positive isolates were serotyped using group-specific antisera (Difco) for somatic factors 4, 5, and 9.

Colorimetric DNA hybridization assay. GN broths (Difco) were inoculated from the same tetra- thionate broths as those used for CB, and assayed according to the instructions provided by the manu- facturer (GENE-TRAK Systems, Framingham, Mass.). Briefly, the GN broth cultures were incubated at 35 C for 6 hr. then they were treated with alkaline lysis solution, which releases the target nucleic acids. After neutralization, DNA probes were added that are ca- pable of hybridizing specifically with Salmonella rRNA. One of these is tailed with poly deoxyadenylic acid, whereas the other is labeled with a fluorescein, acting as a hapten in a later detection step. A plastic dipstick coated with long polymers of deoxythymi- dinylic acid is then added. The probe-target complex binds to the dipstick, which is then reacted with en- zyme-labeled anti-fluorescein. Color was generated by exposing the dipstick to a substrate-chromogen mixture, and its intensity was compared to that of both positive and negative controls included with the kit.

Statistical analyses. Data were analyzed by the contingency chi-square or analysis of variance meth- ods using the Minitab? statistical software package (Minitab Inc. State College, Pa.).

RESULTS

The duration of infection was measured by determining if Salmonella could be identified from cloacal swabs taken on three postchal- lenge occasions (Table 1). Both CB and GT- DNAH methods were applied to each specimen by using the tetrathionate broths as the source of material for further assay. Thus the data pre- sented in Table 1 represent the comparative ca- pability of the two methods (CB vs. GT-DNAH) to detect any Salmonella present at the point of selective-enrichment. Because there were no statistically significant differences in recoveries (chi-square 1 df = 0.65, 0.5 > P > 0.75) be- tween hens challenged with both Salmonella and the Proteus sp. versus those challenged with Salmonella alone, these results were com- bined.

Although there was no statistically significant difference between the two assay methods in detecting Salmonella based on the total num- ber of cloacal recoveries (26 by CB and 37 by GT-DNAH), the latter method was numerically superior at each sample occasion (chi-square, 1 df = 2.63, 0.75 > P > 0.90). Cloacal recovery of S. enteritidis was consistently higher from the white leghorns compared with the brown egg producers (chi-square, 1 df = 12.9, P < 0.001).

The total egg production, the number of de- fective eggs, and the number of eggs from which bacteria other than Salmonella could be isolat- ed are given in Table 2. There were no statis- tically significant differences between brown versus white egg types for total egg production, or the number of defective eggs (ANOVA, F = 3.97, P= 0.08; and F= 2.51, P= 0.15, respec- tively). Egg defects included, in order of fre- quency, elongated shape, thin shells, off-white

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P. F. Cotter et al.

Table 2. Total and defective egg production and bacteriological test results from experimental hens

cloacally challenged with S. enteritidis.

No. No. Hen No. No. Salmonella bacteria type No. eggs defective % assayed % positive positive

White 490A 116B 24 102 21 0 5C Brown 570 77 13 139 24 0 6

AANOVA total egg production, F= 3.97, P= 0.08. BANOVA defective egg production, F= 2.51, P= 0.15. CTen isolates were Proteus sp., one was not identified.

color (tints), small size, wrinkles, and pimples. Both the data for total egg production and for the production of defective eggs were also ex- amined to determine if there was an effect that could be ascribed to the presence of Proteus

sp. None could be detected (ANOVA, F= 0.01, P= 0.9; and F= 1.5, P= 0.25, respectively). There was a highly significant effect on egg pro- duction and the production of defective eggs based on the number of days postchallenge, however (ANOVA, F= 19.8, P= 0.0; and F= 8.5, P = 0.007, respectively). The hen-day av-

erages reached their peak values of 12.2 and 11.6 on postchallenge day 28 for the brown and white hens, respectively. Those same values had declined to 0.8 and 4.6, respectively, by the ter- mination of the experiment on day 50 (Fig. 1).

Selection of eggs for bacteriologic testing was based on their appearance; those showing poor shell quality (defects) were preferred, whereas

10-

I 5

0

* o WSP(10) o + WS(10)

x BSP(10) 6 * BS(9)

x

9 x

+

9

10 20 30 DPC

40 50 40 50

Fig. 1. Egg production (HDA = hen-day averages; DPC = days postchallenge) in a commercial strain of white leghorn hens (W) or a brown egg-producing strain (B) after a cloacal challenge of S. enteritidis alone (S), or combined with a Proteus sp. (SP). The parentheses contain the number in each group.

the few with gross fecal contamination were excluded. Additional assays where performed on some normal-appearing eggs collected from hens having a history of a positive cloacal swab

sample. No Salmonella were identified from any of

the eggs by either assay method; however, other bacteria were recovered from 11 of 241 (4.6%) eggs assayed. Ten of these isolates were iden- tified as Proteus sp. using CB. Nine of the 10 Proteusisolates came from hens challenged with Salmonella enteritidis plus Proteus sp., but it was not determined if these isolates were iden- tical to the inoculated species. The remaining isolate was not identified.

Gross necropsy results performed on all ex-

perimental hens on day 50 are given in Table 3. Tissue samples from the ovary, ceca, and co- lon were removed aseptically and assayed for the presence of Salmonella using CB only. All results were negative. However, the tetrathion- ate broths of two of the colon specimens taken from white leghorns became suspiciously clouded after prolonged (>7 day) incubation at room temperature. Salmonella were subse- quently identified from both using GT-DNAH. One of these specimens was obtained from a hen with a history of two previous positive clo- acal swab samples, and the other from a hen with no prior positive cloacal samples.

DISCUSSION

The results of the present study agree with our earlier observations that showed that GT- DNAH is comparable with CB in detecting Sal- monella from cloacal samples (9,10). Although more samples were found to be positive using the GT-DNAH method, the difference was not significant. Moreover, there was no instance in which a specimen found positive by CB was not

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S. enteritidis identification by DNA hybridization

also found positive by the GT-DNAH method.

Alternately, there were nine cloacal samples identified as positive using GT-DNAH from which S. enteritidis was not identified by CB. Although speed was not considered in the ex- perimental design, our observations imply that the potentially more rapid GT-DNAH method offers some advantage in obtaining results, while not sacrificing the sensitivity of CB.

The apparent infection rate declined from a

high of 19 of 39 total hens (49%) on postchal- lenge day 14 to a low of 8 of 39 (20%) by day 42 as shown in Table 1. A similar decline in the

apparent infection rate was observed by us ear- lier (9,10) and was found by Shivaprasad et al. (11) who observed fecal shedding for 17-21

days, depending on the trial, after intracloacal inoculations of commercial white leghorns. Moreover, Gast and Beard (6) reported a sig- nificant decline in recoveries after 14 days in

orally challenged hens. Positive tests were more frequently obtained

from the white leghorn strain throughout the

experimental period. Moreover, they produced fewer eggs during the post-challenge period (Fig. 1), a numerically higher proportion of which were defective. Two of the leghorns were found by necropsy to be out of production, and three were found to contain an internal egg (Table 3). Taken together these observations suggest they were more susceptible to the chal- lenge as compared to the brown egg strain.

There were several noteworthy observations regarding the cloacal swab recoveries. First, there were two hens from which a negative sam- ple was bracketed by two positive samples (+, -, +). There was a single example of a positive sample occurring only at the last sample (-, -, +), and an additional case of three negative cloacal samples but with a positive necropsy sample from colon tissue. Such observations are difficult to interpret but might be due to the transient presence (intermittent shedding) of the bacteria in the colon, or they may reflect deficiencies in either the sampling techniques (the use of a swab) or the detection methods.

The occurrence of the delayed positive sam- ples from two tetrathionate broths, as men- tioned above, suggests that on occasion the number of viable bacteria in a sample is initially very small. Reaching the minimal level required for detection may not be possible in the time usually allotted to either the pre-enrichment or

Table 3. Gross necropsy observations and conven- tional bacteriology results from hens cloacally chal- lenged by S. enteritidis obtained at postchallenge day 50.

No. of No. of Salmonella Hen observationsA isolates Hen type No. NVL FL OP IE Ceca Colon Ovary

White 20 11 1 2 3 0 0/2B 0 Brown 19 13 5 0 1 NEC 0 NE

ANVL = no visceral lesions; FL = fatty liver; OP = out of production; IE = internal egg.

BPositive by GT-DNAH only after prolonged incu- bation at room temperature.

CNE = not examined.

selective enrichment phases of the assay meth- ods.

The production of eggs having various shell defects after cloacal exposure to S. enteritidis was reported in our earlier study (9,10) and was repeated in the present study. Salmonella en- teritidis could not be recovered from any of the 193 defective or 50 normal eggs tested by either assay method, however. The presence of four internal eggs detected at necropsy (Table 3) may explain in part the production of shell ab- normalities. Their presence in the abdomen may cause an unusual pressure to be exerted on oth- er eggs during their time in the shell gland. Internal eggs were detected at necropsy in our earlier study, some of which were positive for S. enteritidis, others were not. Moreover, in one instance, 13 normal-appearing salmonella-neg- ative eggs were recovered from a single hen in which a salmonella-positive internal egg was detected at necropsy (9,10). Misshapen ovules, and egg peritonitis were reported by Hopper and Mawer (7) from hens identified epidemi- ologically as the source of raw eggs that caused an outbreak of human food poisoning. No men- tion of shell defects was made in that report, nor were such defects reported by Lister (8) who found similar lesions in a broiler flock pos- itive for S. enteritidis.

Ten eggs were positive for the presence of Proteus sp. and one additional unidentified iso- late was made from another egg using CB. These could have been fecal contaminants, but the low (11/1060, 1%) level of bacteria in the eggs suggests that our method of including the shell along with the internal contents does not result in gross bacterial contamination of the sample.

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Our earlier experience (10) in isolating Pro- teus sp. from eggs prompted the decision to include it in half of the challenge inoculations. However, there was no statistically detectable effect on any of the experimental parameters due to the use of the mixed inoculum.

In conclusion, our present results support our earlier demonstration of the usefulness of the GT-DNAH method in detecting Salmonella in cloacal samples taken from poultry. This meth- od seems to be at least as sensitive as the CB method, and offers a potential advantage in the time needed to complete the assay. Moreover, the colorimetric procedure is free of any dis- advantage associated with the requirement for a radioactive probe. Our observations with ex- perimental Salmonella enteritidis infection in laying hens may be of interest to those inves- tigating this emerging human health problem (1).

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11. Shivaprasad, H. L., J. F. Tmoney, S. Morales, B. Lucio, and R. C. Baker. Pathogenesis of Salmonella enteritidis infection on laying chickens. I. Studies on egg transmission, clinical signs, fecal shedding and serological response. Avian Dis. 34:548-557. 1990.

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