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EPIDEMIOLOGY
Surveillance in livestock and poultry feed. Swabs were taken from the surfaces and abdominal cavities of 300 chicken carcasses from 20 flocks in Portugal during 1986-1987. Of these 171 (57%) were Salmonella positive, 6 of which carried 2 serotypes. Serotypes isolated; were enteritidis (66%); agona (12%); newport (6%); saintpaul (6%); derby (4%), typhimurium (3%), bardo (1%) ohio (1%), untypable (2%). S. enteritidis was found in every flock. The results are compared with those of avian and human Salmonellosis registered in Portugal during the same period. (Machado-J and Bernado-F, 1990). The Ministry of Agriculture, Fisheries and Food has released the (MAFF, 1996 Report) on Salmonella in livestock and feed. The poultry breeding flocks and hatcheries (PBFH) order of 1993 was introduced to comply with European Union Directive 92-117 EEC which mandates procedures and frequency of salmonella monitoring. In terms of the MAFF nomenclature, a Salmonella "incident" represents the first and all subsequent isolations of a specific serotype from a farm hatchery, plant or feedmill. "Isolation" represents the recovery and identification of a Salmonella SPP-from a single specimen or submission. During Calendar 1996, 915 incidents were recorded in chickens, of which 60% were attributed to routine surveillance. Approximately 16% were derived from diagnostic activities and 12% were associated with the PBFH order. A total of 231 incidents were documented in turkey with the majority arising from diagnostic activities and surveillance in farms and hatcheries represented the majority of incidents associated with chickens (97%) and turkeys (95%). Collectively, commercial chickens, turkeys and ducks represented 30% of the 4,140 incidents from all Livestock species during 1996. Salmonella enteritidis remained the principle serotype isolated from chickens in 1996. Of the 962 incidents, 177 (18%) were attributed to this serotype. Improved control measures have reduced the number of Salmonella incidents and the relative importance of S. enteritidis during the past five years. By comparison in 1990, 843 of 1906 Salmonella incidents (19%) were associated with S. enteritidis. Consistent with previous years, S. enteritidis phage type 4 was most common accounting for 64% of the 161 incidents where phage type was determined. Salmonella ohio (11%), S. typhimurium (9%) and S. senftenburg (8%) were the most frequent isolates from chickens. Among turkeys, S. typhimurium (30%) and S. newport (24%)were the most frequently encountered isolates among the 231 incidents of Salmonellosis documented on farms and in hatcheries. Seventy-four percent of the S. typhimurium isolates from turkeys corresponded to type 104. Game birds remain an important reservoir of Salmonellosis. It is considered significant that 7% of the incidents in this ranges of species included S. pullorum (7%) and S. enteritidis (12%). Biosecurity procedures to reduce the prevalence of Salmonella infection in commercial poultry should incorporate strict measures to separate commercial and free range game birds. Isolates of Salmonella sp. From processing plants and points of sale reflected the prevalence of S. enteritidis and S. typhimurium in the live population. Forty percent of the 46 Salmonella incidents involved S. enteritidis. It is encouraging that S. enteritidis and S. typhimurium are now relatively rare in feed ingredients. These pathogens were isolated from only 6 and 36 samples respectively from 71,000 ingredient submissions which were examined during 1996. It is noted that from the end of March 1996 all meat and bone meal derived from cattle and sheep was excluded from U.K Livestock feed. Accordingly surveillance of domestic and imported vegetable proteins will be intensified in the future. Salmonella senftenburg, S. ohio and S. mbandeka were the top three isolates from poultry feed in 1996. Approximately 2% of 8870 samples of pelleted poultry feed yielded Salmonella in 1996, confirming that processing meal by pelleting and extrusion does not completely eliminate Salmonella infection. The MAFF reported a 44% recovery of Salmonella from 5712 samples of "pig and poultry meals" reflecting the low recovery rate in ingredients and the imperfect inhibitory effect of pelletization. Animal protein sources represent the highest potential for introduction of Salmonella sp. into the integrated food chain.During 1996 13% of 13 domestic batches were positive and 26% of 19 samples of white fish meal yielded Salmonella. Contamination rates of imported protein ingredients were much higher than the domestic products Approximately 25% of 814 samples of either meat, meat and bonemeal or greaves were positive. The imported proteins were collectively associated with an average 14% Salmonella recovery rate compared to 9% for equivalent domestic products. Approximately 50 % of 1314 salmonella isolates were sensitive to 16 antimicrobials. The highest levels of resistance was noted against sulfonamides (37%), tetracyclines (24%) streptomycin (25%) and ampicillin(24%). There is no evidence that antimicrobial sensitivity has increased from 1996. It was determined that 11% of stock and feed has resulted in a reduction in recovery rates in live birds and products . The prevalence of salmonella infection in UK livestock is a reflection of the intensity of surveillance. Reviewing data assembled by MAFF since 1993 indicates that incidence rates are responding to control measures despite the growth and intensification of the industry. The following figures were taken from ZOOTECNICA International, December 1997. PP 6-10 :
In an epidemiological survey of fowl diseases in Shaonxi (China), Salmonellosis was identified as one of the infective diseases which seriously affected the poultry industry. (Wang-Jingxu et, al ,1996) Among the 23 Salmonella strains isolated from 21 diseased chickens and 2 embryonated eggs in 6 poultry farms near Lusaka city, Zambia, Serovars identified were gallinarum (11 strains), Virginia (1 strain), Haifa (1 strain) and Dublin (1 strain). S. gallinarum was detected at the highest incidence from all inspected farms. 4 serovars have been reported for the chickens in Zambia so far. Alamo, Hifa and Virginia were identified for the first time in this study (Tuchili-LM et al., 1996a) According to the quarterly report of the United States Department of Agriculture National Veterinary Services Laboratory (USDA-NVSL,1996 report), S. heidelberg was the most frequently isolated serotype (Figure 5) from poultry. The "other" category in chickens included 3 isolate of S. enteritidis. A wider range of Salmonella serotypes was isolated from turkeys as shown in figure 6. The "other" category included 3 isolates each of S. arizonae, S. heidelberg and S. javiana. The following figures were taken from ZOOTECNICA International, October 1996:pp 16. Figure 5: Salmonella serotypes (Isolates from clinical cases) most frequently identified in chicken flocks. Figure 6: Salmonella serotypes (Isolates from clinical cases) most identified in Turkey flocks. Salmonellosis was recorded as one of the poultry diseases diagnosed in Zaria between January 1986 and December 1995 and was more prevalent in poultry above 12 weeks old (Halle -PD et, al 1998). Caecal contents of 2345 broiler chickens from 12 farms (28 flocks) in Japan, collected between April 1995 and March 1997, were examined for Salmonella. Salmonella was isolated from 336 (14.3%) samples, eight serovars were identified. 242 isolates (72.0%) were identified as S. blockley, 69 (17.9%) as S. hadar, 15 (4.5%) as S. bredency, 9 (2.7%) as S. Schwarzengrund, 4 (1.2%) as S. anatum, 3 (0.9%) as S. enteritidis, ;2 (0.6%) as S. ohio, and 1 (0.3%) as S. livingstone. The same serovars were repeatedly found in chickens from the same farms. S. typhimurium and S. ebterutudus were detected in pooled broken eggshell samples collected from the hatchery. Analysis of plasmid profiles revealed 11 patterns of S. blockley and 7 patterns of S. hadar. S. blockley strains with the same plasmid profiles were isolated repeatedly from the same farm for over a year. (Limawongprance-S et al., 1999). Many enteric diseases cause problems in today's poultry industry around the world, with the most significant ones being coccidiosis, Fowl cholera , Malabsorption syndrome (MAS), Nectrotic enteritis, enteric parasites and the various effects following an infection with Salmonella. Salmonella, Campylobacter and E. coli infections continue to emerge as being important discussion points, perhaps influenced by the implication of the risk of being potentially involved in human food poisoning. These potential threats for human health are often caused by poor sanitation , bio- security and poor methods of disinfection in hatcheries, farms and processing plants (Wiebe van dir Sluis, 1999). Occurrence in poultry Meat and Eggs The level of Salmonella contamination in retail chicken products was investigated bimonthly in two countries in the UK over 12 consecutive months. In total 325 chicken and 35 giblet samples were tested. Supermarket chicken, fresh chilled and frozen, and chicken from local butcher shops were compared for their rates of Salmonella contamination which were, respectively, 18.6, 25.5 and 24.5% with an overall rate of 22.8%. Giblets were contaminated at a rate of; 37.1%. Amongst all Salmonella isolated, Salmonella enteritidis was present in 51.4% and S. typhimurium in 12.2% of the chicken samples and 23.1 and 0% respectively of the giblet samples (plummer-RAS et.al., 1995) In Olsztyn, polland, 37,779,959 carcasses and internal organs of slaughtered fattened poultry (hens, chickens and turkeys) were examined during 6 years, 1986- 1991. 1.66% of the birds examined were condemned for human consumption. The percentage of condemned birds was highest for hens (2.4%) and the lowest in chickens (1.27%) Salmonellosis was diagnosed in 13463 chickens (0.056%), 15951 hens (0.94%) and in 3918 Turkeys (0.032%) (Radkowski-M et al., 1996). Dressed chickens from markets in Metro Manila, Philippines, were tested for salmonella between November 1989 and 1990. Swabbing was done on the skin surface of the brest, inner thigh and perianal region. Identification of the organism was based on cultural and biochemical characteristics and by slide agglutination test using polyvalent antigen - O serum. 20% of the chickens sampled were positive for salmonella. The isolation rates did not differ significantly between chicken dressed within market premises.(Baldrias-LR and Capistrano-RVS, 1997). Salmonellosis is one of the commonly recorded diseases with various degree of severity in the presented data on poultry diseases collected at various veterinary institutions in Serbia between 1989 and 1998 (Palic-T et al., 1998). During 1993 to 1996, a study was conducted to isolated and characterize Salmonella in poultry carcasses and their products sold in Belgium. The rate of contamination increased from 19.4% in 1993 to 36.7% in 1996. A 100% increase of Salmonella positive samples resulted from cutting up the carcasses into individual parts. Chicken parts were more often contaminated with Salmonella than turkey poults. The predominant 3 serotypes were S. enteretidis (16.3% ) - S. hadar(15.5%) and S. virchow (14.1%). S. newport was frequently isolated from turkey products. (Uyttendaele-MR et al., 1998). Salmonella Prevalence in crop contents Swabs of crop contents of 635 broiler chickens from 9 ontaria and 12 quebec Processing plants in Canada were cultured for salmonella. Serotypes of positive cultures were determined. The overall prevalence of contamination was low (4.3%), 5.6% in Quebec and 2.2% in ontorco. The serotype profiles of isolated salmonella was similar to profiles obtained from other surveys of Canadian broiler flocks using either environmental samples or cloacal swabs. Similarities included predominance of S. hadar and S. Heidelberg; several other common serotypes at a low prevalence; little s. enteritides isolated in other studies, and no s. enteritidis isolated in this study.(Chambers-JR. et al., 1998). Prevalence in free-range Ducks and hens and in wild birds and Ostrich In 1992-1993,16 serotypes of Salmonella were identified from traditional hatcheries. Dead embryos, moribund ducks, drinking water, feed , dust and faeces were collected for examination, and 191 Salmonella isolates were obtained. All the isolates were confirmed and serotyped. The 16 serotypes were hadar (27.7%), typhimurium (24.6%) omsterdam (10.5%), virchow (7.8%) Seneftenberg (7.3%), Thompson (6.8%) paratyphi B Var Java (3.7%), Ouakamaa (3.1%), weltevreden (2.1%), sofia (1.6%), group B (1.1%) Oslo (1.1%), Lexington (0.5%), group E and C (0.5%), derby (0.5%) and agona (0.5%), (Istiana, 1993). A total of 2983 sparrows, 22 batches, from chicken farms in the south of Taiwan were examined for S. pullorum and Mycoplasma by serum plate agglutination tests and cultured for isolation of Salmonella spp. and Mycoplasma spp. Seven isolates of Salmonella SPP included S. welteverden, S. agona, S. london, S. infants, and for the first time in Taiwan , S. azteca. 11.6% of examined sparrows were positive for pullorum. (Lin-MY et al., 1996). One hundred eggs from free range hens and 100 eggs from free range ducks collected from local markets during winter of 1995 were examined for Salmonella at the Salmonella National Center, Baghdad, Iraq. S. typhimurium was isolated from 6 hen egg shells and 6 duck egg-shells. S. montevideo was isolated from 2 duck egg shells.(shareef -AM et al 1997) In Indonesia between 1992 and 1993, 22 isolates of S. hadar were isolated from 180 internal organs of ducklings (12.2%). One isolate of S. typhimurium (0.5%) and one isolate of S. Virchow (0.5%) were isolated from 180 duck's eggs. No pathogenic bacteria were isolated from feed or rice bran (Suryana and Istiana, 1997). Salmonellosis was one of the most frequent primary cause of death in (0.7%) of 386 wild birds submitted to the Quebec centre, for wild Animal Health, the Quebec region of the Canadian cooperative Wildlife Health Centre, and the laboratories of the Quebec Ministry of Agriculture, fisheries and food between 1993 and 1996. (Mikaelian-I, 1997). Only 1 (1%) of 124 cloacal swabs from free-flying avian species yielded salmonella sp. compared with 21 (17%) samples positive for Campylobacter SP. Of 171 racing pigeons from 8 owners, 8 (5%) yielded Salmonella spp. all of which were serotype typhimurium. It is suggested that although the prevalence of Salmonella and Campylobacter in wild life in Trinidad were low, wildlife farming and increased consumption of meat from wildlife may increase the risk of zoonosis (Adesiyun-AA et.al, 1998). Thirty percent of the Ostrich farms in Compaiar, Italy were visited. Ostriches were housed differently from between 3 and 40 breeding birds. Only some farms organized the complete management of eggs; their collection, Storage and incubation. The clinical findings reported were due to infectious diseases and to different pathologies related to incorrect management. Salmonellosis was one of the most frequently identified infectious diseases in ostriches. (Cerrone-A et al., 1999). Incidence and Impact of Salmonella in Broiler Hatcheries Cox-N (1999) reported that: For many decades commercial feed and ingredients were thought to be the primary contributors to Salmonella contamination of poultry. Most poultry producers believed that if Salmonella could be eliminated from feed, the problem will be solved. Many studies have shown that Salmonella serotypes on the fully processed broiler carcass originate from sources other than feed. These include hatcheries, breeder flocks and litter. The poultry industry in the USA incubates over eight billion broiler hatching eggs each year. Bacteria, including Salmonella, can rapidly penetrate the shell and membranes of the freshly laid fertile hatching egg. The conditions that exist during the incubation of a hatching egg tend to favor the proliferation of these microorganisms. Invading Salmonella do not usually cause extensive decomposition of the fertile egg and the chicks hatches, resulting in the creation of Salmonella reservoirs in commercial broiler and breeder hatcheries and broiler populations. These reservoirs will continue to exist until Salmonella have been eliminated from broiler breeder flocks or until Salmonella are eliminated from fertile eggs by a suitable chemical treatment. The surface of most fertile hatching eggs can be effectively disinfected with an effective chemical applied as soon as possible after exposure of the egg to Salmonella. Once microorganisms such as Salmonella penetrate membranes of hatching eggs there is no effective way to remove, kill or prevent their further invasion of the egg contents and developing embryo. After incubation, Salmonella trapped in the membranes can be ingested by the embryo at the time of internal piping or as it emerges from the egg. In addition during the process of hatching, one contaminated chick can causes the spread of Salmonella to most of the other chicks in the hatching cabinet due to the circulation of air by fans. Exposure of newly hatched chicks to Salmonella is extremely critical because the young chick lacks a mature gut microflora and is highly susceptible to intestinal colonization. Research has shown that very low levels of Salmonella can colonize the intestinal tract of young broiler chicks through the mouth, eye, nasal passage or navel. This can potentially result in the production of shedders. A chick that become colonized in the hatchery can subsequently spread Salmonella contamination to other chicks both within the hatchery and to flock-mates during grow-out. When an infected flock reaches the processing plant, Salmonella contamination, both intestinal and environmental, can be released into the processing plant environment and contaminate the final product from that and subsequently processed flocks. Data from several field trials has been used to track Salmonella serotypes originating from the hatchery to the final processed carcass. Competitive exclusion treatments have been shown to protect chicks from low level challenge by environmental Salmonella. Pre-exposure to Salmonella in the hatchery can reduce the effectiveness of treatment. In a large scale commercial field trial, the Salmonella serotypes recovered from processed carcasses of the competitive exclusion treated flocks were the same as those serotypes isolated from the hatchery samples. In these trials, some of the chicks were colonized with Salmonella from the hatchery before the competitive exclusion treatment could be administered. As a result, this early exposure to Salmonella limited the effectiveness of the competitive exclusion treatment. On a positive note, the Salmonella contamination in US broiler hatcheries has been dramatically reduced since 1990. These impressive reductions were most probably influenced by one or a combination of the following:-
The Role of defeathering in carcass contamination A study was undertaken to determine whether the incidence of either Salmonella spp. Or listeria monocytogenes on turkeys at 3 commercial processors could be related to the type of defeathering system: 1\rsb\ conventional, 58 degree C. Common bath scald; 2\rsb\ Kosher, 57 degree C common bath scald, 3\rsb\ steam-spray, or 62 degree C nonimmersion scald. Flocks were sampled before defeathering, after defeathering, and after chill at each facility. The incidence of Salmonella-positive turkeys significantly increased subsequent to conventional defeathering (10 positive out of 14) as compared with before defeathering (3/14). The number of Salmonella-positive carcasses following Kosher (0/14) and steam-spray (2/14) defeathering were similar to the number of Salmonella-positive carcasses found prior to defeathering (1/14 and 3/14) respectively. The incidence of Salmonella-positive carcasses following chill was slightly lower, but not; significantly different than the number of Salmonella-positive carcasses found immediately following defeathering at all processes. (Clouser-CS et.al, 1995a). The cross-contamination effects of 3 commercial defeathering systems were compared using turkeys from a single salmonella-positive flock (15% cloacal-positive). Single or "common" flocks were used to control flock-to-flock variability. Thirty birds were mechanically defeathered in each system as the first flock of the day and compared with 30 hand-defeathered (control) birds. Three trials, each using a different common flock, were completed. Results were recorded. It is hypothesized that the increase in the number of Salmonella-positive birds following steam-spray and Kosher defeathering in trials 2 and 3 were a result of skin surface changes occurring during the defeathering process, which allowed increased adherence or entrapment of Salmonella spp. On or within remaining skin layers. (Clouser-CS et.al., 1995b). USAHA Web-1998 Report of the USAHA Committee In Calendar year 1997, there were three isolations/ outbreaks of S. pullorum reported to the poultry improvement staff, two classic strains and one variant. There was one isolation of S. pullorum reported during calendar year 1998 from January to October 1st, 1998. Isolations in 1997 were reported by 3 states. In 1998, two states reported isolates and the two isolates were standard strain. There have been no isolations of S. gallinarum since 1988. Most producers of eggs in the USA are concerned about the effect of Salmonella enteritidis (SE) in their flocks on food safety and are using some form of SE control program official or unofficial. During the last year, outbreaks of human disease traced back to layer flocks occurred in the Midwest resulting in the initiation of a state of Quality Assurance program for monitoring and controlling SE. The route of spread of SE to different farms is thought to possibly be due to contaminated egg flats that were not properly washed, use of contaminated spent fowl equipment to move pullets, and contaminated flies on trucks. Serotyping results of Salmonella from animals and related sources reported during July 1997 to June 1998 on 19,438 Salmonella isolates from animals and epidemiologically related sources were reported. The most frequently identified serotypes were S. typhimurium, S. heidelberg, S. senftenberg, S. Kentucky, and S. derby. Twenty-three percent of the total isolates were S. typhimurium and it was among the 10 most frequently identified serotypes from chickens, turkey, cattle, swine and horses. Salmonella bredency and S. mbandaka were included among the 10 most frequently identified serotypes for the first time. In addition 1857 isolates of S. typhimurium and s. typhimurium var Copenhagen were tested for resistance to ampicillin, chloramphenical, streptomycin, sulfonamides and tetracycline. Of the isolates tested, 27% were resistant to the five antibiotics tested (penta-resistant). Sixty-two percent of these isolates were also resistant to trimethopirm. Sixty six percent of penta-resistant isolates were s. typhimurium var Copenhagen. Penta-resistant isolates were received from 33 states and one foreign country. The most common phage among the penta-resistant isolates was DT 104 (45%) followed by DT1046 (17%) In an update from the Animal protein producers industry (APPI) on the incidence of Salmonella in protein meal products; in 1998, 22.5% of submitted samples were identified as positive for Salmonella. The particular serotypes isolated from a small sample of animal protein meals included, s. senftenberg, S. bredency, S. mbanadaka, and S. montevideo. The APPI monitoring programe has provided substantial benefits to the rendering industry. The workers concluded that a number of routes of entry of Salmonella are possible in highly susceptible, newly-hatched chicks. These became seeders, disseminating infection by the fecal route. Preharvest control of Salmonella infection will depend on enhancing hygiene in breeder houses and suppressing infection in the hatchery. Given the complexity of intensive broiler production, restrictions on the use of certain disinfectants including formalin as a fumigant and the high levels of mechanization in hatcheries, integrators can only hope to suppress Salmonella infection.(USAHA Web, 1998). Salmonella sp. Colonization Various routes of contamination of day old broiler chicks, with a nalidixic acid-resistant strain of S. typhimurium was evaluated by workers at the USDA, Russel research center. Inoculation by oral gavage, ocular, and aerosol routes resulted in high levels (80-100%) cecal colonization at 6 days of age. Application of the organism to the navel of young chicks was a less effective route of infection although some replicates were colonized (USAHA Web, 1998). Groups of 10 newly hatched chicks were inoculated orally with antibiotic-sensitive acid-resistant strains. 24 h later they were incubated with nalidixic acid-resistant strains. The inhibition of colonization of the chicken caeca with nalididixic acid-resistant strains varied considerably. Some strains were much more inhibitory than others. The most inhibitory strain, S. typhimurium strain maidstone inhibited 6 of the 12 strains tested whereas the least inhibitory, S. kentucky, inhibited only one strain. There was not an absolute relationship between inhibitory activity and colonization ability. No relationship was seen between inhibition and serotype or phage types within serotypes. There was no correlation between in vivo inhibition and the extent of inhibition that occurred in early stationery phase cultures in rich, undefined broth cultures. (Iba-AM et.al., 1995). Experimental infection Experimental infections of different salmonella serotypes were established in a commercial line of ducks to provide baseline information for control measures. The ducks were very resistant to systemic infection with S. typhimurium, S. enteritidis and S. gallinarum within 36 h of hatching. This was associated with an inherent inability of the strains to multiply in the reticulo-endothelial system. The resistance was not associated with poor invasiveness or serum sensitivity. Individual strains of S. typhimurium, S. enteritidis, S. heidelberg and S. orion colonized the gut well and were excreted in the faeces for at least 6 weeks by ducks when they were infected orally within 2 days of hatching. The main sites of colonization were the caeca and, to a lesser extent, the crop. Viable counts of each inoculated strain in the caeca remained in excess of 106 c.f.u. 3 weeks after infection although the organisms had been cleared from the spleen by this time. Much less excretion occurred when the birds were infected at 3 weeks of age. When infected ducks, which had cleared the infection, were challenged orally with the homologous strain expressing a different genetic marker, very low levels of excretion of the challenge strain were detected when compared with a control group. After infection low titres of circulating LPS-specific IgG antibodies were detected by an ELISA. Intestinal colonization of newly hatched ducks with an aroA strain of S. enteritidis resulted in extensive colonization which exerted an exclusion effect on the parent strain inoculated 24 h later (Barrow-PA et al., 1999). ETIOLOGY Classification and nomenclature (cited by Calnek-BW et al., 1997) The genus Salmonella is a member of the bacterial family Enterobacteriaceae and is divided into five biochemically distinct subgenera. The various motile and non-host-adapted serotypes of subgenus I are often referred to as paratyphoid(PT) salmonellae. The degree of genetic relatedness among the Salmonellae is so great that some researchers suggested that the genus actually consists of only a single species, but the names of individual serotypes remain in common usage to facilitate diagnostic classification and epidemiologic analysis. Salmonella pullorum and S. gallinarum are both highly host-adapted and among the few members of the genus Salmonella that are nonmotile and they belong to the serogroup D according to the Kauffman White Scheme. Morphology and staining The organisms are gram-negative, nonsporogenic, motile or nonmotile rods measuring about 0.7-1.5x2.0-5.0 um. The bacilli mostly occur singly , but occasionally two or more can be found united. Typical Salmonella colonies on agar media are about 2-4 mm in diameter, round with smooth edges, slightly raised, and glistening (cited by Calnek-BW et al., 1997). Isolation and cultural characteristics Malmann et al., (1928) isolated S. pullorum from feces Of dead chickens using brilliant green liver infusion agar. The liver was the most constant site of gross lesions in chickens infected with S. pullorum followed by lung, heart, gizzard and caeca (Doyle and Mathews 1928) Malmann(1929) recommended tetrathionate broth, selenite F broth, S.S agar and MacConkey agar for Salmonella isolation. Gwatkin and Glover (1930) isolated S. pullorum from the nasal passages of fowls. Gwatkin (1946) isolated S. gallinarum-pullorum from heart, lung and liver of 39 out of 59 experimentally infected young chicks.Cattow (1951) studied the efficacy of some selective media for salmonella isolation from chickens. Fragments of liver, lung, intestinal mucosa and its contents were incubated for 18 hours at 37 C in tetrathionate broth. Loopfuls were then plated on MacConkey's agar medium, bismuth sulphite medium, S.S agar medium and incubated for further 24 hours at 37 C . Cook et al., (1951) reported that selenite F broth was better than tetrathionate broth as an enrichment medium and plating from selenite F broth onto desoxycholate citrate agar was the most successful method. Nitzschke (1951) concluded that tetrathionate broth is better than selenite F broth if salmonella is accompanied by E. coli. Harvey and Thomson (1953) found that 43 C is the optimum incubation temperature for selenite F broth cultures for isolating Salmonellae. Smith and Williams (1954) isolated the organism from trachea and lung of orally infected chicks. Beattie (1960) reported that bacteriological examination of yolk material from embryos dying from 19-21 days of incubation proved a practical method for detection of Salmonella infection. McCoy (1962) found that incubation of tetrathionate broth at 43 C was lethal to Salmonella and most of other microorganisms. Bulling and Pietzsch (1966) found that potassium tetrathionate broth was slightly superior to selenite F broth and that brilliant green phenol red lactose agar was superior to Gassner medium for the isolation of salmonella from poultry. Bassioni and Farid(1967) found that the most suitable media for salmonella isolation from poultry organs were Gassner's metachrome yellow lactose agar, Endo's fuchin lactose agar, MacConkey's bile salt neutral red lactose agar respectively proceeded by enriching on tetrathionate broth which was found to be superior to selenite F broth, when this microorganisms were accompanied with E. coli. Harvey and Price (1967) recommended the incubation of Selenite F broth at 43 C as enrichment for Salmonella isolation, then subculturing on brilliant green agar incubated at the same temperature for 48 hours to reduce the number of Proteus organisms. Greenfield and Bankier (1969) concluded that selenite F. broth incubated at 43 c for 48 hours was most favorable method for salmonella isolation. Geissler and Kosters (1970) found that a large proportion of S. gallinarum-pullorum strains, incubated at 43 C in tetrathionate broth, showed a poorer growth on solid media than after incubation at 37 C. The authors conclude that, although enrichment at 43 C inhibits contaminants, it may be injurious to fastidious salmonella serotypes and should be employed only in addition to incubation at 37 C. Greenfield and Bigland (1970) studied the effect of incubation temperature of selenite F broth on several salmonellae and found that S. cholera-suis, S. gallinarum-pullorum and S. typhimurium did not grow well specially when incubated at 42 C rather than 37 C. Smyser et al., (1970) compare between 3 enrichment media incubated at 42 + 1 C for isolating Salmonellae. The media used were selenite brilliant green sulfapyridine, dulcitol selenite sulfapyridine and tetrathionate brilliant green. They concluded that each media incubated at elevated temperature was significantly more effective than tetrathionate brilliant green incubated at 37 C for isolation salmonellae. Carlson and Snoeyenbos (1974) reported that tetrathionate was generally better than selenite F broth regardless of the sample to be cultured at either 37 C or 42 C after 24 hours or 48 hours. S. pullorum grew well on bismuth sulphite and MacConkey's agar but did not grow on S.S agar or brilliant green agar. Elsawy (1976) concluded at selenite F broth was superior to tetrathionate broth in isolating salmonella from ducks. He found that the optimum temperature was 43 C for Selenite F broth and 37 C for tetrathionate. Moreover S.S agar was found by him to be superior to MacConkey's agar for salmonella isolation. Siddique and Bercca (1984) studied the efficacy of some enrichment media and selective media for isolating salmonella gallinarum, they found that the best results from direct plating of organ suspensions were obtained with Desoxycholate citrate lactose agar followed by MacConkey's agar and S.S. agar. Detection of salmonella in poultry, meat, and environment Detection by Drag-swab sampling and Salmonella antigen-capture technology: Drag swab (DS) sampling, at the rate of 4 DS gauze pads/flock; modified culture procedures (novobiocin-supplemented plating media and delayed secondary selective enrichment); and salmonella antigen-capture (SAC) technology were combined in screening one layer flock and 38 market-age broiler flocks. The results showed that low (negative) SAC sample/positive (S/P) ratios were related to the negative culture recovery of salmonella. Similarly, high (positive) S/P ratios were related to and indicative of positive culture recoveries. Extensive sampling and testing of 18 of the 39 flocks disclosed :A) that five flocks with negative culture recoveries from feathers and freshly voided feces had essentially no positive DS-SAC values and B) that 13 flocks with positive culture recoveries from feathers and/or fresh feces all had positive DS-SAC values (mallinson et al., 1989) Detection by sandwich ELISA: A system of detecting a wide range of Salmonella serotypes, based on a single non-selective culture followed by a sandwich ELISA showed 80% agreement between conventional microbiological procedure in an inter-laboratory trial involving 3 laboratories. It is concluded that the specificity of the monoclonal antibodies replaced the conventional enrichment broth and offered a considerable time saving. (Wyatt-GM et.al. 1995) Detection by Dot-blot ELISA using Monoclonal antibodies: Monoclonal antibodies (MAB) produced to polysaceharides in the LPS molecule of Salmonella were used in a dot-blot ELISA for detecting Salmonella in 873 food samples including 100 fresh chicken, 261 frozen chicken, 100 hen eggs and 100 duck eggs. Salmonella culture from foods involved the following steps, pre-enrichment, enrichment in selective medium, isolation or; selective and complete indicator media, followed by biochemical and serological identification of appropriate colonies respectively.The whole culture procedures of dot-blot ELISA took < 2 hours. Salmonella could be recovered from 16% of fresh chicken, 8.8% of Frozen chicken and 2% each of hen eggs and duck eggs. S. paratyphi B biovar Java was predominated in chicken. The MAB-based dot-blot ELISA were positive in 30% of fresh chicken, 27.6% of frozen chicken, 2% of hen eggs and 4 % of duck eggs. The sensitivity and specificity of the MAB- based dot-blot ELISA compared to the bacterial culture method were 81.5% and 85% respectively. The discrepancy of the data between the culture method and the dot-blot ELISA might be due to the fact that the culture method could detect only living cells at numbers that gave at least one isolated colony on the selective differential media plate while the dot-blot ELISA detects any form of Salmonella antigen. The MAB-based dot-blot ELISA offers several advantages over the conventional bacterial culture method when it is used for screening of Salmonella contamination in foods, especially export foods. This include rapidity, Cost-effectiveness and simplicity (the dot - blot ELISA does not need highly trained personnel or equipment, in contrast to the cultural method). The test can be performed in field conditions and the result can be read visually. It also offers multi sample analysis at one time, which renders more samples of food for screening possible, thus false negative results are fewer which, in turn, assures the quality of the export food in a cost-saving, short time frame (Chaicumpa-w et al., 1995). Detection by immunomagnetic separation: Immunomagnetic separation techniques were used in the isolation of salmonella from raw chicken carcasses. Improved isolation rates were achieved with increased specifically and decreased processing time, although several technical difficulties remain to be addressed. Immunomagnetic separation offers significant potential for improvement of existing microbiological systems for the isolation of salmonella (Coleman-DJ et al., 1995). Detection by DNA probe: A standardized method for the identification of Salmonella SP. Using a specific insertion sequence (IS-200) of Salmonella chromosomal DNA as a probe. Colony hybridization was positive for 10 Salmonella strs of various serotypes and negative for 18 bacteria of the family Enterobacteriaceae. Sensitivity of detection was 1-6 c.f.u. using 32p.-labeled probe. Chicken (16 samples) and fish (6 samples) were tested using the IS-200 DNA probe after pre-enrichment and selective enrichment. All samples tested positive with the major stress identified as S. gallinarum, S. typhimurium, S. enteritidis, S. choleraesuis and S. paratyphi (Bandekar-JR et al., 1995). Detection by polymerase chain reaction (PCR): PCR was used after a short pre-enrichment culture to detect Salmonella subspecies in chicken fillets. A direct PCR assay performed with chicken meat inoculated with S. typhimurium produced no PCR products. Six different DNA extraction protocols were tested to recover efficiently Salmonella DNA after a short incubation period. Three of them gave results that were reliable, rapid and sensitive. Successful protocols used proteinase K and/or a centrifugation step to concentrate the samples. For reliable detection of Salmonella subspecies, a few thousand bacterial cells per ml must be present. To obtain this number of bacterial cells with an inoculation of about one cell in 25 g of ionized food products, required 8 h in culture broth to give positive results by PCR-based assay. (Soumet-C et.al-1994). A multiplex PCR technique was compared with standard bacteriological techniques for the detection of Salmonella, and S. enteritidis in particular, in samples of chicken skin obtained from retail outlets-five of 68 samples were positive by both systems (7.4%), 58 of 68 were negative by both systems (85%), 5 were positive by PCR only (7.4%) but none were positive by culture only. The PCR was quicker and more sensitive than the bacteriological methods, finding 15% of samples positive for Salmonella compared with 7.4%, and taking < 24 h to reach a result as opposed to 2-3 days by culture methods (Mahon-j et.al, 1994). By PCR using a pair of primers specific for Salmonella phoE gene, a 365-bp specific gene fragment could be amplified from yolk of infertile eggs and dead-in-shell chicken embryos, and from environmental samples. 44.4% of dead-in-shell embryo samples were positive for Salmonella DNA by PCR compared with 24.4% by bacteria isolation. Salmonella DNA could also be detected in infertile eggs, chicken feces, floor litter and chick fluff (Tuchili-LM, 1996b). BAX, a commercial PCR-based system, was evaluated to determine the efficacy of the system with different concentrations of Salmonella cells in mixed cultures and compared to conventional methods for detection of inoculated Salmonella cells from poultry samples. When present in enrichment broth at levels of 105, 104, and 103 C.F.U./ml, Salmonella cells were detected in 23 of 150 (15%) processed chicken rinse samples with the BAX system compared to 18 of 150(12%) samples with conventional methods. Salmonella cells were detected in 28 of 50 (56%) ground turkey samples with the BAX system compared to 25 of 50 (50%) samples with conventional methods. Overall, there was a 97% method agreement. One false-negative and 2 false positive results were obtained with the BAX. Band sizes and intensity of the PCR product were shown to be correlated with estimated numbers of Salmonella (C.F.U) present in the enriched and inoculated samples. The assay was able to detect 104 Salmonella C.F.U per 1 ml of enriched medium, allowing consistent detection of Salmonella cells within 24 to 26 h. It is concluded that the high degree of sensitivity and specificity of the BAX system make it a reliable alternative to conventional bacterial culture methods (Bailey-JS, 1998). Detection of Salmonella in food with 1-2 tester: Food samples of different origin including poultry meat, turkey meat and egg salad were investigated to compare a recent commercially available immunodiffusion 1-2 tester (Bio control) with a cultural standard method (non-selective pre-enrichment in buffered peptone water, selective enrichment in Rappaport-Vassiliadis-medium and selenite brilliant-green mannitol enrichment broth, inoculation on 2 selective agars) for presence of Salmonella. The 1-2 tester showed in positive cases an accuracy (sensitivity) of 94.9% and is therefore considered suitable for detection of Salmonella contaminated food, (Weber-A and Rachelmann-E, 1991) Detection by stabilized bismuth sulphite agar (DurapakTM): The combination of a new manufacturing process, packaging, and media engineering has contributed to the development of extended shelf-life (stabilized)- plated media (Durapak TM). One of the first products stabilized by this process was bismuth sulfite agar plates (BSA). Stabilized BSA were stored 3-4 months at 35 degree C and then were inoculated in parallel with laboratory-prepared BSA following overnight aging at room temp. Several strs of Salmonella were tested in pure cultures and in foods, including raw ground turkey, chicken, and nonfat dry milk. Also, two naturally contaminated raw ground turkey and chicken samples were tested. Both lab-prepared and stabilized BSA detected all positive samples after 24 h incubation at 35 degree C. Recovery, colony size and amount of sheen produced by Salmonella were generally equivalent on both types of BSA, but stabilized BSA often yielded enhanced blackening of colonies and of the surround medium. Results were dependent upon strain of Salmonella used, type of food and type of enrichment broth streak onto the plates. Stabilized BSA were generally more inhibitory to Salmonella than lab-prepared BSA, as demonstrated with pure cultures and food samples. Overall, the performance of stabilized BSA was equivalent to that of lab-prepared BSA. (Allen-SB, et al., 1993) Detection by the Tecrea R uniqueTM Salmonella Test: One hundred samples consisting of chicken carcass rinses, chicken parts, dust samples from chicken barns and mechanically separated turkey meat were tested for Salmonella by pre enrichment in modified semisolid Rappaport Vassiliadis (MSRV)medium. Thirty-six percent of the samples were presumptively positive for Salmonella with Tecra R test of which one was a false positive result. Fifty-nine percent were positive by selective migration in the MSRV medium. The Tecra R uniqueTM Salmonella test had a sensitivity of 59.3% and a specificity of 97.6% compared with the MSRV method. (Poppe-C and Duncan-CL, 1996). Identification & Characterization By Conventional and rapid biochemical tests: Of the 29 outbreaks of Salmonella investigated in Turkish between October, 1993, and September, 1994, 21 were in Commercial layers, 2 in broilers and 1 each in a hatchery, breeder layers, a broiler abattoir, turkeys, pigeons, and quails of the isolates, 16 were S. gallinarum, 9 S. enteritidis and 4 S. typhimurium. The isolates were identified by conventional and rapid biochemical tests. In 156 suspected colonies the agreement between results was 98% with the lysine decarboxylase test (with 3 negative results), 99% with the urease test (with false positives), 100% with indole test, and 98% with the malonate test (with 3 negative results). Most of the isolates were resistant to erythromycin but sensitive to fluroquinolones. (ozdemir-U, 1996). Calnek-BW et al.( 1997) cited that: The biochemical properties characteristic of most PT (subgenus I) Salmonella strains are described by Krieg and Holt (1984) and Ewing (1986) Typical PT Salmonellae ferment glucose(to produce both acid and gas), dulcitol, mannitol, maltose and mucate but do not ferment lactose, sucrose, malonate, or salicin. They can produce hydrogen sulphide on many types of media, decarboxylate ornithine and lysine, utilize citrate as a sole source of carbon, and reduce nitrates to nitrites. Paratyphoid Salmonellae do not hydrolyse urea or gelatin and do not produce Indole. Paratyphoid Salmonellae can be distinguished from S. arizonae (Salmonella subgenus III), S. pullorum and S. gallinarum on the basis of several biochemical differences. For example, S. arizonae strains cannot ferment dulcitol but usually can ferment malonate, S. pullorum strains cannot ferment mucate or dulcitol, and S. gallinarum strains cannot decarboxylate orinthine or produce gas from glucose fermentation. In addition, PT Salmonellae are usually motile but S. pullorum and S. gallinarum are nonmotile. There are more similarities than differences between S. pullorum and S. gallinarum in their biochemical properties. Both organisms can ferment arabinose, dextrose, galactose, mannitol, mannose, rhamnose, and xylose to produce acid, with or without gas production. Substances not fermented include lactose, sucrose, and salicin. One important biochemical difference between these two organisms is that S. gallinarum ferments dulcitol, whereas S. pullorum does not. Also, S. pullorum only occasionally ferments maltose. The major difference between the two organisms is, however, that S. pullorum cultures produce rapid decarboxylation of ornithine, whereas cultures of S. gallinarum do not. In addition, S. gallinarum uses citrate, D(-)sorbitol, L(-)fucose, D(-)tartrate, and cysteine hydrochloride gelatin. Some of these differences are helpful in differentiating the two organisms; however, variation in the behavior in some strains can occasionally be observed, especially in regard to gas production. Ribotyping by the use of the enzyme EcoRI has also been suggested as an important tool to differentiate between S. pullorum and S.gallinarum. Genetic Characterization: Twenty Salmonella strains (13 S. enteritidis, 3 S. typhimurium, 2 S. derby and 2 S. gallinarum pullorum) isolated from 10 poultry farms (in Romania) were characterized by ribotyping, IS typing and RAPD (PCR random variant). S. typhimurium strains were differentiated from the other strains by all the techniques. S. gallinarum-pollurum and S. enteritidis strains were discriminated by RAPD; ribotyping, however, demonstrated similarities among the 2 serovars suggesting gene rearrangements, as well as differences among S. enteritidis strains within the same RAPD profile. S. derby was not differentiated from S. gallinarum-pullorum and S. enteritidis by any of the methods (Danes-D et. al, 1995). Resistogram typing of Salmonella serotypes: The resistogram typing of the 65 Salmonella isolates of poultry and its environment has shown 36 different patterns. Among the various dyes and chemicals used, organisms showed a high rate of resistance to the chemicals such as 0.7% copper sulphate (81.54%) and 0.003% malachite green ( 78.46%) followed by 0.09% acriflavine (69.23%) and 0.0006% phenyl mercuric nitrate (67.69%). The copper sulphate and malachite green have the least discriminating ability in the differentiation of strains in resistogram typing. Among the dyes and chemicals used only 3 out of 65 isolates showed resistance to 0.7% 4 chloresorcinol. (Purushothaman-V et.al, 1998). Mutation Rate The rate at which resistance developed in avian coliform bacteria when exposed to nalidixic acid, Sarafloxacin or enrofloxacin was investigated. In invitro studies, the rates of mutation of avian isolates of E-Coli and Salmonella were measured following nalidixic acid, sarafloxacin or enrofloxacin exposure. The rates of mutation were similar for nalidixic acid and sarafloxacin, whereas a lower rate of mutation was seen after enrofloxacin exposure. In-vivo, the quinolones were given in drinking water to broilers at 40 ppm for 5 consequitive days. Samples of feces were inoculated onto appropriate media and the frequency of resistance was measured. The frequency rates of resistance to nalidixic acid and sarafloxacin were similar. Enrofloxacin-medicated birds did not develop enrofloxacin resistant coliform bacteria. The invitro and in-vivo results appear to be correlated. (Medders-WM et. al, 1998).
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