Of Salmonellae and Salmonella Gozo LORANNE VELLA* Introduction had to carry out the study, I realized that Gozo was just small enough and had just the right I will always remember the summer of 1993 as number of cattle farms for my project. There a particularly hot and humid season, not to were other advantages: my father could help mention my experiences chasing, with gloved to take me around the various farms for the hands and booted feet, a few hundred samples (since I had no car) and I could speak apprehensive and distrustful cows for some the various Gozitan dialects, which made the stinking, fresh dung. The memories now are farmers more receptive. quite hilarious, but not then, at least not when the bulls or a rather angry cow had to be confronted. The Genus Salmonella Having always been rather keen on the study Salmonella is a group of bacteria consisting of of micro-organisms, I knew right from my more than 2000 different types (known as freshman year at the University of Malta, that serotypes or serovars); these are all potentially I wanted to work in a topic related to pathogenic to man and may cause microbiology for my undergraduate thesis. In gastroenteritis, septicaemia or enteric fever. 1991, I spent a whole summer reading Medline Salmonellae also infect many animal species, abstracts, but it was an article on Salmonella in including birds and reptiles. In man, infections one of my Medicine Digest copies that actually often result following ingestion of improperly caught my attention. Another reason that cooked animal products that have been encouraged me to undertake a study on previously contaminated with faecal matter Salmonella was the fact that no such studies on during processing. This results in an acute, self- animals had been carried out before in Malta. limiting gastroenteritis1 or a systemic infection I remember writing a brief protocol about what (enteric fever) involving other areas of the body. I wanted to do, going to the Bacteriology Patients with acute gastroenteritis excrete large laboratory in St. Luke’s Hospital and asking numbers of bacteria in faeces but numbers Dr. PauI Cuschieri, the Consultant diminish as recovery progresses, so that after 3 Bacteriologist in charge of that laboratory, if he to 4 weeks, stools are usually bacteria free. could be my tutor and if what I had in mind However, some patients continue to excrete was feasible. Happily enough, he was quite salmonellae for a longer period, which ranges interested and accepted without hesitation. So from months to several years, although started a long and eventful study on permanent chronic carriage lasting for more salmonellae. Initially, I wanted to study than one year is very rare. The case fatality rate salmonella carriage, infection and disease in is low, usually less than 0.4% and deaths mainly poultry but complications arose, primarily occur in the very young, the very old and in because permission to visit poultry processing debilitated, immuno-compromised persons. plants was almost impossible to obtain. Next, The generic term Salmonella was given to the I focused my attention on pigs; however, I micro-organism in 1900 in honor of Dr. D. E. practically knew no one in Malta who could Salmon (1850-1914), an American pathologist take me around the various pig farms in Malta. who, together with Theobald Smith, was the In the end, keeping in mind the limited time I first to discover and describe Salmonella. On * Ms Loranne Vella is a pharmasist. She is currently working the surface of this bacterium, there are various as a mycologist. She was responsible for the setting up of proteins called antigens, which differ in their the first mycology laboratory in the Maltese Islands. nature between the different salmonellae. This 1 The gastrointestinal form is often referred to as food enables scientists to distinguish between one poisoning, although this is a misnomer, since the disease is an infection rather than an intoxication. Salmonella serovar and another; thus, Salmonella THE GOZO OBSERVER (No.12) - March 2005 12 Enteritidis has different antigens on its surfaces nine consecutive weeks from each individual compared to say, Salmonella Montevideo. animal chosen from 52 farms distributed in 12 Historically, salmonellae have been classified towns in Gozo. The 300 bovines sampled according to their antigenic structure (i.e. the represented 12.5% of the total bovine types of antigens present) and each new type population of Gozo whilst the 52 farms that is discovered is named after the place in sampled constituted 83.9% of the total available which it was first isolated. The Kauffmann- sampling points (farms = 62). Each swab was White scheme is a systematic tabulation of the placed in a special medium to protect it, stored different antigenic structures of the Salmonella at 4ºC whilst in transit and processed in the serovars known. In this classification system, laboratory within 24 hours of collection. the genus Salmonella (abbreviated to S.) is Isolation and identification of the Salmonella divided into two species, namely, S. enterica isolates were carried out using standard and S. bongori. Salmonella enterica is further procedures. Antibiotic sensitivity testing on subdivided into 6 subspecies, the most the isolated strains was performed using the important of which is Salmonella enterica impregnated disc diffusion method (Stokes’ subspecies enterica. Most of the Salmonella modification of the comparative method); the serovars pathogenic for mammals and birds antibiotics chosen included ampicillin, belong to this subspecies, although members cefuroxime, chloramphenicol, ciprofloxacin, of S. enterica subspecies arizonae are important furazolidone, gentamicin, sulphafurazole, causes of diarrhoeal illness in turkeys and are tetracycline and trimethoprim. Throughout therefore of worldwide economic importance. the study adequate internal quality control was carried out including quality control on culture media used for the isolation of Salmonella The Study serovars, quality control during the actual isolation and identification of the organisms, In 1993, I therefore started a cross-sectional controls during serological testing and controls study on a representative sample of cows and used for the antibiotic sensitivity testing. bulls from various farms in Gozo, the main aims of which were: Results and Discussion 1. to determine the prevalence of Salmonella excretion/carriage in these Of the 300 faecal samples taken, 41.3% (n = 124) animals; yielded Salmonella and more than one serovar 2. to identify the most common serovar were isolated from seven swabs; hence, there prevalent within the study population; were 131 isolates in all, which increased the and, percentage of Salmonella isolated to 43.7%. 3. to determine the presence of any Since a single faecal swab was taken from each serovar resistance to the antibiotics animal, this means that 41.3% of the animals commonly used in animal husbandry. sampled were excreting the organism at the time of sampling with 7 of the animals The study population consisted of 2325 cows excreting more than one type of Salmonella. and 68 bulls. A representative sample of 300 head was selected using strict random The cows and bulls yielding positive Salmonella sampling from 62 farms spread in 14 towns in cultures did not show any clinical signs of Gozo. These consisted of 296 cows and 4 bulls; salmonellosis such as fever, dullness, the animals were from 3 to 4 years old and the diminished appetite, blood-stained faeces and cows consisted of both wet (milked) and dry in the case of cows, reduced milk yields. Since (non milked) animals. Calves and one year old the animals were excreting the organism, it is heifers and bulls were excluded. more likely that they were Salmonella carriers rather than cases of acute infection. However, Faecal swabs were obtained every week for from this study, it cannot be concluded 13 THE GOZO OBSERVER (No.12) - March 2005 whether this was temporary or permanent However, lack of reporting of individual cases carriage. The nature of carriage is particularly especially if mild or no symptoms are important because it affects the persistence of experienced have to be kept in mind. It could Salmonella in the herds. Additionally, cattle also be due to the fact that the serovars isolated from Gozo are used for human consumption from the cattle studied have a low both in Malta and Gozo. This increases the pathogenicity and are easily destroyed by opportunity of Salmonella dissemination at the proper cooking. abattoir prior to slaughter with a consequent increase in the risk of meat contamination. Salmonella serotype Antigenic structure No. of Isolates Croft 28: g,m,s: e,n,z15 55 (18.3%) Telaviv 28: y: e,n,z15 29 (9.7%) Montevideo 6,7: g,m,s: 1,2,7 17 (5.7%) Gozo 28: e,h: e,n,z15 14 (4.7%) Kpeme 28: e,h: 1,7 7 (2.3%) Infantis 6,7: r: 1,5 2 (0.7%) Abadina 28: g,m: e,n,z15 1 (0.3%) Other* - 6 (2.0%) Total 131 (43.7%) * These were rough strains that could not be serotyped and hence, identified; consequently, they cannot be classified under any serogroup. Table 1: Number and types of Salmonella serovars The New Salmonella Serovar Grum stain of Salmonella showing Grum-negative rods (x1000) A total of 14 Salmonella isolates belonged to a serovar that was subsequently confirmed to be Seven different kinds of serovars were isolated, a new serovar by the WHO Collaborating including a new serovar for which the name Centre for Reference and Research on Salmonella Salmonella Gozo was given (Table 1). When in France. All isolates were biochemically taken together, the three serovars S. Croft, S. typical of the Salmonella genus. This isolate Telaviv and S. Montevideo, constituted up to could not be serotyped easily with the 33.7% of the total number of samples taken.