Because E. coli is ubiquitous in the intestinal tract of mammals and can be isolated from every faecal specimen, diagnosis of K99 infection requires the detection of strains carrying the K99 antigen from the faeces of the affected calves. The definitive diagnosis of K99 ETEC infection relies on the demonstration of this fimbrial antigen in the E. coli strain isolated from the faeces (Holland, 1990). Briefly, following isolation of an E. coli from a faecal specimen, suspected K99 positive colonies are grown on specific selective media that enable expression of the K99 antigen (Acres, 1985), and this can be identified by agglutination with specific antisera. In order to circumvent the isolation phase ELISA tests for the diagnosis of the K99 antigen directly from faeces have been developed using monospecific fimbrial antisera
28 (Nagy and Fekete, 1999). Thanks to their versatility and the possibility of production of polyvalent kits covering BRV, BCV and Cryptosporidium antigens (Izzo et al., 2011; Jäkel, 1995; Schneider et al., 1984), numerous commercial ELISA kits for detection of K99 antigen are currently available, and to the author’s knowledge ELISA kit technology has replaced bacterial isolation methods for K99 in most New Zealand diagnostic laboratories.
Under natural conditions, adequate colostral immunoglobulin transfer may reduce the severity of K99 diarrhoea. However, calves may remain susceptible to K99 infection within the first few days of life, even if adequately fed colostrum after birth. Therefore, specific immunising agents against the K99 antigen are often necessary (Radostits, 1991; Snodgrass et al., 1982). As in the case of BRV and BCV, passive immunity obtained by vaccinating pregnant cows during the last trimester of pregnancy with K99 antigen can be obtained. Vaccines are usually commercially prepared using inactivated K99 pili antigen, and are administered subcutaneously to healthy pregnant cows at two doses, the first six to seven weeks before parturition, and the second dose three weeks later (Nagy, 1980; Nagy and Fekete, 2005; Walker et al., 2007). This approach decreases the severity of the infection and diarrhoea but it does not completely prevent the clinical disease (Nagy, 1980; Snodgrass et al., 1982). Following the same rationale, an alternative passive immunisation via oral administration of specific antibodies obtained from hyperimmune chicken egg yolk IgY or bovine colostrum has been reported. This strategy, which has been explored during the past two decades, could represent a new, promising, economically convenient approach to the prevention of gastrointestinal infections in neonatal calves (Ikemori et al., 1992; Mine and Kovacs-Nolan, 2002; Vega et al., 2011). In a trial conducted by Ikemori et al (1992), the protective results of egg yolk IgY obtained from chickens immunised with heat-extracted K99 antigens were estimated in an experimental calf model of K99-induced diarrhoea. In this experiment, the results showed that young calves fed milk containing egg yolk powder with specific IgY had temporary diarrhoea, 100% calf survival and better body weight gain, compared to the control calves that suffered from severe diarrhoea and died within 72 hours after infection.
29 2.3.2.4. Infection with Salmonella spp.
Salmonellosis is an infectious disease of humans and animals caused by bacteria belonging to the genus Salmonella. Salmonella spp. cause diarrhoeal and systemic infections in humans. The organisms are commonly found in subclinically infected farm animals, leading to contamination of animal products (meat, milk and eggs). Other sources of human infection are fruits and vegetables irrigated or fertilised with contaminated faecal wastes (Berger et al., 2010; Crum-Cianflone, 2008; Pui et al., 2011).
Salmonella spp. belong to thefamily Enterobacteriaceae. Taxonomically, Salmonella is divided into 2 species; S. bongori and S. enterica. S. enterica is further divided into 6 subspecies: S. enterica subsp. enterica (I), S. enterica subsp. salamae (II), S. enterica subsp. arizonae (IIIa), S. enterica subsp. diarizonae (IIIb), S. enterica subsp. houtenae (IV) and S. enterica subsp. indica (VI) (Grimont and Weill, 2007). According to the WHO Collaborating Centre for Reference and Research on Salmonella in France, the total number of Salmonella serotypes recorded in 2006 was 2579 (Grimont and Weill, 2007). As the ‘official’ nomenclature is very long, in the literature salmonellae are commonly named using abbreviations. For instance, Salmonella enterica subspecies enterica serotype Typhimurium is abbreviated as S. Typhimurium (Grimont and Weill, 2007).
Salmonellosis has been documented worldwide (World Health Organization Global Salm-Surv, 2006) and most cases of salmonellosis in humans and animals are caused by serotypes of Salmonella enterica subspecies enterica. The organisms infect most species of domestic animals, especially pigs, calves and poultry (Wray and Wray, 2000). Enteric disease is the most common clinical presentation, but a wide range of forms, such as acute septicaemia, abortion, and arthritis may be seen (Wray and Wray, 2000). In addition, several host species, particularly pigs and birds, may be subclinically infected. Subclinically infected animals play an important role in Salmonella transmission within and between herds, and as sources of foodborne infection in humans (Hendriksen et al., 2011; Lo Fo Wong et al., 2002; Pui et al., 2011).
In calves, salmonellosis is often transmitted from subclinical infected cattle, birds or contaminated feed (Anderson et al., 2001). A wide range of Salmonella serotypes
30 have been reported in newborn calves (Anderson et al., 2001; Izzo et al., 2011; Langoni et al., 2004; Reynolds et al., 1986). Infection severity and mortality rates in young calves generally reflect the strength of immunity (Anderson et al., 2001). In the calf, immunity is enhanced by good colostrum management (Vermunt et al., 1995; Wesselink et al., 1999) and the provision of adequate nutrition, as well as optimal environment conditions that minimise pathogen load and bacterial challenge dose through contaminated milk and equipment (Mohler et al., 2009).
Salmonella spp. are identified as Gram-negative, rod shaped, highly motile organisms (with the exception of S. Gallinarum and S. Pullorum which are non-motile). Biochemically, Salmonellae are catalase-positive, oxidase-negative, non-lactose fermenting, indole -negative, urea- negative, methyl red and Simmons citrate - positive and H2S producing (Russell and Gould, 2003). Salmonella has a complex life cycle in the infected host, and multiple virulence genes which enable pathogenic Salmonella to invade the host’s intestinal epithelium and macrophages have been identified (Marcus et al., 2000). An important step in the pathogenesis of Salmonella infection is the bacterial interaction with the reticuloendothelial system of the host, which promotes inflammation (Chopra et al., 1999; Francis et al., 1993; Galán, 1996). Further, the capability of some Salmonella spp. tosurvive and replicate inside macrophages promotes apoptosis, and this allows evasion from the host’s immune response (Aranda et al., 1992; Chopra et al., 1999; Rathman et al., 1996). Salmonella virulence genes are often clustered in pathogenicity islands, which are usually absent in non-pathogenic strains (Blanc-Potard et al., 1999). According to Marcus et al., (2000), these pathogenicity islands are classified into five subgroups labelled as SPI- 1 through SPI-5, from which virulence genes SPI-1 and SPI-2 have higher virulence.