Protective immunity conferred by a DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine when delivered in-water to sheep challenged with Salmonella enterica serovar Typhimurium.

Stimulation of acquired immunity to Salmonella in livestock is not feasible in neonates (which can be infected within 24h of birth) and is challenging in feedlots, which typically source animals from diverse locations and vendors. Induction of innate immune mechanisms through mass vaccination of animals upon arrival to feedlots is an alternative approach. Transport, environmental conditions, changes in social grouping, and further handling during feedlot assembly are significant stressors. These factors, as well as concurrent exposure to a diversity of pathogens, contribute to the risk of disease. We have shown that oral immunization of calves with a modified live Salmonella enterica serovar Typhimurium vaccine strain, which lacks the DNA adenine methylase gene (S. Typhimurium dam), attenuates the severity of clinical disease, reduces fecal shedding, and promotes clearance of salmonellae following virulent homologous and heterologous challenge. This study examines the safety and efficacy of a S. Typhimurium dam vaccine in sheep via oral delivery in drinking water (ad libitum), as a means to effectively vaccinate large groups of animals. Adult merino sheep were vaccinated in drinking water -28 days, -7 days and 24h pre and 24h post-virulent Salmonella Typhimurium challenge which was administered via the oral route. Significant attenuation of clinical disease (temperature, appetite, and attitude) and reduction in mortality and virulent Salmonella Typhimurium fecal shedding and tissue colonization was observed in animals that received the vaccine 28 and 7 days pre-challenge. Further, vaccination did not pose a risk to stock previously infected with virulent salmonellae as mortalities and clinical disease in sheep vaccinated prior to or following virulent challenge did not differ significantly from the non-vaccinated controls. The capacity of S. Typhimurium dam vaccines delivered in drinking water to protect livestock from virulent Salmonella challenge offers an effective, economical, stressor free Salmonella prophylaxis for intensive livestock production systems.

Cross-protective immunity conferred by a DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine in calves challenged with Salmonella serovar Newport.

Intensive livestock production and management systems are associated with increased fecal-oral pathogen transmission and a resultant high prevalence of multiple Salmonella serovars in many large dairy farms and feedlots. Thus, it is imperative to develop livestock vaccines that are capable of eliciting potent states of cross-protective immunity against a diversity of serovars of a given species. Immunization with modified live Salmonella enterica serovar Typhimurium vaccine strains, that lack the DNA adenine methylase (Dam), confers cross-protective immunity in murine and avian models of typhoid fever as well as in a bovine model of salmonellosis. Here we examined whether a dam mutant Typhimurium vaccine (serogroup B) has the capacity to elicit cross-protection against a virulent challenge with an emerging, clinically relevant, and multi-drug resistant strain of serovar Newport (serogroup C2-C3) that has been associated with clinical disease in recent salmonellosis outbreaks in calves. Vaccinated animals challenged with Newport exhibited a significant attenuation of clinical disease (improved attitude scores, increased daily weight gains and reduced fever and diarrhea) and a concomitant reduction in Newport fecal shedding and colonization of mesenteric lymph nodes and lungs compared to non-vaccinated control animals. The capacity to elicit cross-protective immunity in calves suggests that dam mutant vaccines have potential application toward the prevention and control of Salmonella infection in commercial livestock production systems wherein livestock are exposed to a diversity of Salmonella serovars.

Salmonella and on-farm risk factors in healthy slaughter-age cattle and sheep in eastern Australia.

OBJECTIVE: To examine healthy slaughter-age cattle and sheep on-farm for the excretion of Salmonella serovars in faeces and to identify possible risk factors using a questionnaire. PROCEDURE: The study involved 215 herds and flocks in the four eastern states of Australia, 56 with prior history of salmonellosis. Production systems examined included pasture beef cattle, feedlot beef cattle, dairy cattle, prime lambs and mutton sheep and animals were all at slaughter age. From each herd or flock, 25 animals were sampled and the samples pooled for Salmonella culture. All Salmonella isolated were serotyped and any Salmonella Typhimurium isolates were phage typed. Questionnaires on each production system, prepared in Epi Info 6.04, were designed to identify risk factors associated with Salmonella spp excretion, with separate questionnaires designed for each production system. RESULTS: Salmonellae were identified in all production systems and were more commonly isolated from dairies and beef feedlots than other systems. Statistical analysis revealed that dairy cattle were significantly more likely to shed Salmonella in faeces than pasture beef cattle, mutton sheep and prime lambs (P<0.05). A wide diversity of Salmonella serovars, all of which have been isolated from humans in Australia, was identified in both cattle and sheep. Analysis of the questionnaires showed access to new arrivals was a significant risk factor for Salmonella excretion on dairy properties. For beef feedlots, the presence of large numbers of flies in the feedlot pens or around stored manure were significant risk factors for Salmonella excretion. CONCLUSION: Dairy cattle pose the highest risk of all the slaughter-age animals tested. Some of the identified risk factors can be overcome by improved management practices, especially in relation to hygiene.

Passive immunisation of neonatal lambs via colostrum and milk of ewes previously immunised with live attenuated Salmonella typhimurium protects neonatal lambs from experimental salmonellosis.

Lambs sucking non-immunised ewes or ewes immunised 4-5 weeks before lambing with live attenuated, aromatic-dependent (aroA) Salmonella typhimurium (strain CS 332) were challenged orally at either 2, 4 or 7 days of age with virulent S. typhimurium (strain CS 94) at doses ranging from 10(9) to 10(13) colony forming units. No lambs displayed signs of clinical salmonellosis and all survived challenge but those sucking immunised ewes had organisms of the challenge strain in their faeces for much shorter periods of time than lambs of the control ewes. High titres of specific antibodies were measured in colostrum and milk of immunised ewes in comparison with very low titres measured in samples from control ewes; these differences were reflected by the titres of antibodies in the sera of corresponding lambs. At 2 days after lambing, the major antibody isotype in the colostrum of immunised ewes and sera of their lambs was IgM whereas at 7 days IgG1 was the predominant isotype. While it was clear that vaccination of pregnant ewes with the live attenuated vaccination conferred protection against experimentally-induced salmonellosis in their lambs, considerable protection was observed in control lambs in spite of there being very low titres of antibodies in the mammary secretion of their dams. The latter observation could be related to the presence of contain non-antibody potent bactericidal factors previously described in colostrum and milk.

Systemic and mucosal intestinal antibody response of sheep immunized with aromatic-dependent live or killed Salmonella typhimurium.

Following the development of a suitable formulation capable of inhibiting intestinal proteolytic activity, the total anti-lipopolysaccharide (LPS) and anti-flagellin (Fla) antibody response and isotype in the sera and intestinal washings of sheep, immunized with live aromatic-dependent (aro-) Salmonella typhimurium strain CS332 by the intramuscular (live i.m.) or oral (live oral) route or acetone-killed virulent S. typhimurium by the intramuscular route (killed i.m.), were determined at various intervals post-immunization. The serum or intestinal anti-lipopolysaccharide (LPS) or anti-flagellin (Fla) antibody titres of immunized sheep, regardless of the route of immunization, were significantly greater (P < 0.01) than those of non-immune control sheep. Although significant differences between the serum anti-LPS or anti-Fla antibody titres of sheep in various immunization regimes were observed, they were not consistent for different periods post-immunization. The predominant isotype contributing to serum anti-LPS antibody activity was IgM whereas the serum antiflagellar antibody activity was confined to IgM, IgG1 and IgG2. In either case, the contribution of the IgA antibody isotype was minimal. Antibody activity in the intestinal washings of immunized sheep, regardless of the route of immunization was significantly greater (P < 0.01) than that in non-immune control sheep. However, the titres in sheep immunized with the live S. typhimurium vaccines were significantly greater than those immunized with the killed vaccine. The major anti-LPS or anti-flagellin antibody isotype in the intestinal washings of sheep in the live i.m. or live oral groups was IgM at day 7 post-immunization followed by IgG1 and IgG2 at days 14 and 21 post-immunization, with only a minimal contribution by the IgA antibody isotype. On the other hand, the major antibody isotype in the intestinal washings of sheep immunized with the killed S. typhimurium was IgG1.

Development and duration of protection against salmonellosis in mice and sheep immunised with live aromatic-dependent Salmonella typhimurium.

The aim of this investigation was to determine the development and duration of protection in mice or sheep immunised with aromatic-dependent (aro-) Salmonella typhimurium strain CS332, by either parenteral or oral routes. Immunisation of mice by the intraperitoneal or sheep by the intramuscular routes was found to impart protection against oral challenge with the virulent parent S typhimurium strain CS94 as early as seven days after immunisation. In contrast, when immunisation was carried out by the oral route, protection was not evident until three weeks after immunisation. Regardless of the route of immunisation, mice were still partially protected at three months and were fully susceptible at six months after immunisation. In sheep, protection persisted for six months but not 12 months after immunisation. Only parenterally immunised mice and sheep developed high ELISA and, or, agglutinating antibody titres, and cutaneous delayed-type hypersensitivity (DTH) at three weeks after immunisation. Although both antibody and DTH were detectable three months after immunisation of mice with aro- S typhimurium strain CS332, none was detected at six months. Antibody measured by agglutination and ELISA was detectable six months after immunisation in sheep, although no DTH was evident. At 12 months after immunisation low levels of anti-LPS antibody (measurable by ELISA only) were detected in sheep immunised by the intramuscular route.

Progressive ataxia associated with degenerative thoracic myelopathy in Merino sheep.

A neurological disorder in Merino sheep, characterised clinically by progressive posterior ataxia and microscopically by Wallerian degeneration in thoracic segments of the spinal cord, is described. Animals of both sexes were affected, with the earliest onset of disease being at 5 months of age. Most affected animals died before 2 years of age. The clinical, pathological and epidemiological features suggest that this degenerative thoracic myelopathy is a previously unrecognised entity differing from other reported causes of ataxia in sheep in Australia.

Generation of aromatic-dependent Salmonella havana and evaluation of its immunogenic potential in mice and sheep.

The generation of aromatic-dependent (aro-) Salmonella havana (Group G2, 01, 13, 23) from a smooth wild-type parent strain by transduction with phage P1 is reported. Mice immunized with this live aro- S. havana strain (CS234) by the intraperitoneal (i.p.) route were protected against challenge with wild-type S. havana, whereas those immunized by the oral route were not. Mice immunized with two doses of formalin-killed aro- S. havana by the i.p. route were also unprotected, in spite of high antibody titers. However, only those mice immunized with live aro- S. havana by the i.p. route developed significant delayed-type hypersensitivity. Following i.p. inoculation in mice, the aro- S. havana strain CS234 was detected in the liver, spleen and mesenteric lymph nodes on day 9 but not on day 15 post-inoculation (p.i.). On the other hand, when mice were inoculated with the parent wild-type strain (CS4) or the aro- derivative strain CS234 by the oral route, the organisms were recovered from the mesenteric lymph nodes and intestine only on day 3 but not on day 6 post-inoculation. In sheep inoculated with the aro- strain CS234 in the gastroc muscle, organisms were recovered from the muscle, and popliteal and medial iliac lymph nodes for up to 21 but not 28 days p.i. However, no mutant organisms were recovered from liver, spleen, mesenteric lymph nodes or faeces. In orally-inoculated sheep, the mutant organisms were recovered from the mesenteric lymph nodes, rumen, intestinal contents, and faeces up to 14-21 days post-inoculation but not at 28 days. When sheep immunised with the aro- S. havana strain CS234 by the intramuscular or oral route were challenged with the parent wild-type S. havana strain CS4 by the oral route, the latter strain was detectable in the mesenteric lymph nodes and faeces of immune sheep up to 14 days post-challenge in contrast with the non-immune sheep, where the challenge strain was detectable even at 28 days post-challenge. Only sheep immunized by the intramuscular route developed high antibody levels and delayed-type hypersensitivity.

Genetic manipulation of Salmonella serotype Bovismorbificans to aromatic-dependence and evaluation of its vaccine potential in mice.

The generation of smooth aromatic-dependent Salmonella serotype Bovismorbificans (Group C2, O6, 8) from a smooth wild-type parent strain by transduction with phage P1, and conjugation with Salmonella serotype Typhimurium carrying F'-8gal is described. The smooth aromatic-dependent S. serotype Bovismorbificans was non-lethal for mice at an oral challenge dose of 2 x 10(9) cfu (equivalent to 200 LD50 of the parent, wild-type strain). The safety of the auxotrophic mutant was further substantiated by comparing its multiplication kinetics in vivo with that of its virulent parent organisms. Mice immunised with live, smooth aromatic-dependent S. serotype Bovismorbificans by either the oral or intraperitoneal (i.p.) route were protected against oral challenge with virulent S. serotype Bovismorbificans; the degree of protection was significantly better (p less than 0.05) at a challenge dose of 100 or 200 LD50 in mice receiving two rather than one vaccination. In contrast, mice immunised with three doses of the formalin-killed virulent, parent organisms by the i.p. route were not protected, in spite of high antibody titres. Only those mice immunised with the live, smooth aromatic-dependent S. serotype Bovismorbificans i.p. developed significant (p less than 0.01-0.05) delayed-type hypersensitivity.

Immunizing efficacy of aromatic-dependent Salmonella dublin in mice and calves.

Mice immunized with an aromatic-dependent (aro-) S. dublin strain CS101 by either the intraperitoneal (i.p.) or oral route, were protected against oral challenge with a virulent S. dublin strain CS90, the degree of protection being the greatest when mice had received 3 immunizing doses at weekly intervals. Mice immunized with an aromatic-dependent (aro-) S. typhimurium strain CS332 by the i.p. or oral routes were protected against challenge with virulent S. dublin strain CS90 at 1 or 2 weeks but not at 3 or 4 weeks post-immunization. Mice immunized with 1 dose of aro- S. dublin strain CS101 by the i.p. route developed low levels of lipopolysaccharide (LPS) and flagellin-specific antibody but no delayed-type hypersensitivity (DTH) whereas those immunized with 2 or 3 doses developed significantly higher antibody titres and DTH. In contrast, mice immunized by the oral route developed neither significant antibody response nor DTH. The aro- S. dublin strain CS101 could not be detected beyond day 28 post-inoculation in visceral organs including liver, spleen, mesentery, small intestine, caecum or large intestine of mice inoculated by the i.p. route or in mice inoculated by the oral route with the exception of day 42 post-inoculation. Challenge of mice previously immunized with 3 doses of the aro- S. dublin strain CS101 by the i.p. or oral route with virulent S. dublin strain CS90 resulted in their rapid clearance from the above visceral organs. Calves immunized with the aro- S. dublin strain CS101 by either the intramuscular (i.m.) or oral routes were significantly protected against oral challenge with virulent S. dublin strain CS90. In contrast to the observations in mice, somatic (O) and flagellar (H) antibody titres of calves immunized by either route were negligible as were anti-LPS antibody titres. However, flagellin-specific antibody titres were higher in calves immunized by the i.m. than the oral route. These results indicate that the protection observed in immunized mice or calves against oral challenge with virulent S. dublin was unlikely to have been mediated by humoral salmonella-specific immune mechanism(s).

Evaluation of protection against experimental salmonellosis in sheep immunised with 1 or 2 doses of live aromatic-dependent Salmonella typhimurium.

The minimum number of doses of a live aromatic dependent (aro-) Salmonella typhimurium vaccine strain (SL1479), given by the intramuscular, oral or subcutaneous route required to protect sheep from experimentally-induced clinical salmonellosis, was determined. A significant reduction in mortalities and diarrhoea occurred in those sheep immunised with one or 2 intramuscular doses or 2 subcutaneous doses. On the other hand, sheep immunised with one subcutaneous dose were not protected. Immunisation with one or 2 oral doses also resulted in a significant reduction in mortality, although reduction in the prevalence of severe diarrhoea was less consistent. Sheep immunised with a single intramuscular dose of aro- S. typhimurium developed high levels of serum antibodies and significant delayed-type cutaneous hypersensitivity response to homologous Salmonella lipopolysaccharide and flagellin, whereas those with a single oral dose did not. It was concluded that immunisation of sheep with a single oral or intramuscular dose of live aro- S. typhimurium reduced mortalities and the prevalence of diarrhoea in sheep due to infection with virulent S. typhimurium.

Neurovisceral ceroid-lipofuscinosis in blind Devon cattle.

Ten Devon cattle from a single property were affected with blindness from 14 months of age. Severe retinal degeneration progressing to atrophy was associated with widespread intracellular accumulation of pigment in the retinal ganglion cells, central nervous system and major organs. The pigment was consistent with ceroid-lipofuscin granules on histological, histochemical and ultrastructural examination. Although a familial relationship existed between affected individuals, a pattern of inheritance could not be established by examination of available breeding records. The disease is compared to similar disorders reported in man and other species.

Clinical variations of border disease in sheep according to the source of the inoculum.

Non-cytopathogenic pestivirus obtained from lambs with border disease, with or without nervous signs, was inoculated into pregnant ewes at 57 to 65 days of gestation. Live lambs born to inoculated ewes were clinically identical to the lambs from which virus was obtained, ie, either a hairy birth coat with central nervous system disturbance or a hairy birth coat without central nervous system disturbance.

Pathology in the ovine foetus caused by an ovine pestivirus.

Homogenised tissues or tissue culture supernatant fluid containing a noncytopathic pestivirus obtained from a lamb with a neurologic form of border disease, were inoculated into ewes at different stages of pregnancy. Foetal death occurred in 9 ewes of those inoculated between 19 and 47 days of pregnancy while 3 ewes did not lamb. Eight of the foetuses were aborted between 77 and 132 days of pregnancy; of these 6 were autolysed or mummified and one had arthrogryposis. The one full-term dead lamb had a hairy birth coat and lissencephalic micrencephaly. Foetal death occurred in only 7 of 14 ewes inoculated between 57 and 72 days of pregnancy. Four of these ewes aborted between 77 and 108 days of pregnancy and 3 gave birth to full-term, dead, hairy lambs. The remaining 7 ewes gave birth to live hairy lambs with severe inco-ordination. All lambs carried to term and aborted foetuses or lambs that could be examined had a range of intracranial malformations including focal leucomalacia, micrencephaly, hydranencephaly, porencephaly, lissencephaly and cerebellar hypoplasia. Some lambs also had skeletal abnormalities including arthrogryposis, scoliosis and brachygnathia inferior. The pestivirus isolate used in these trials produced more severe effects on the ovine foetus than previously observed in similar inoculation trials using pestivirus isolates from border disease lambs without nervous signs.

The effect of prostaglandins on in vitro limb cartilage differentiation.

A variety of studies indicate that a key event in limb chondrogenic differentiation is a cellular condensation process during which an intimate cell-cell interaction occurs that triggers cartilage differentiation by elevating cAMP levels. It has recently been demonstrated that when limb mesenchymal cells are subjected to high density monolayer culture under conditions conducive to chondrogenesis, they synthesize several prostaglandins, including PGE2 and prostacyclin, which are important local modulators of cAMP formation in a number of cells and tissues. In the present study, we demonstrate that exogenous PGE2 stimulates the in vitro chondrogenic differentiation of the subridge mesoderm of the embryonic chick limb bud. The stimulatory effect of PGE2 is greatly potentiated by the phosphodiesterase inhibitor, theophylline, suggesting its influence on chondrogenesis is mediated by its ability to increase cAMP levels. The stimulatory effect of PGE2 is dose-dependent and can be detected at a concentration as low as 10(-8)M. PGE1 is just as effective as PGE2 in stimulating in vitro chondrogenesis, whereas PGA1 and PGF1 alpha are less than half as effective. Thromboxane B2 has no effect on chondrogenesis. On the basis of our results, the possibility that endogenous prostaglandins might regulate limb cartilage differentiation by acting as local regulators of cAMP content is discussed.

Experimental production of dermatitis in sheep with Pseudomonas aeruginosa.

The attachment, to sheep skin, for 4 days, of control wool pads saturated with sterile culture medium which contained a bacteriostat, induced only a mild dermatitis, whereas wool pads saturated with Pseudomonas aeruginosa culture induced a subacute dermatitis characterised by scaling, microabscess formation and seropurulent exudate. Changes similar to the latter were observed in skin affected by natural fleece-rot which developed spontaneously after 7 days of artificial wetting and in which P. aeruginosa was the predominant species of bacteria. An exacerbatory, if not causal, role for this organism is suggested in the development of the dermatitis associated with fleece-rot and in the exudation of seropurulent material, a step essential in the development of body strike.