Reproductive performance of sows was improved by administration of a sporing bacillary probiotic (Bacillus subtilis C-3102).

This field study assessed the efficacy of a probiotic based on viable spores of Bacillus subtilis C-3102 (Calsporin; Calpis Co. Ltd., Japan) on the health status and productivity of sows and their litters through 2 full, sequential reproductive cycles from service of the first cycle to weaning of the second cycle. Fifty-six sows were allocated to 2 experimental groups, an untreated control (T1) group and a probiotic-treated (T2) group that received the same basal feed as the T1 group plus the probiotic at an approximate allowance of 30 g/t of feed (3 × 10(5) cfu/g). The offspring of T1 and T2 sows were offered basal and T2 creep feed (3 × 10(5) cfu/g), respectively. Health and zootechnical parameters of sows and piglets were recorded. Feeding the probiotic to sows and piglets resulted in significant benefits, observed in both cycles: 1) improved sow body condition during pregnancy (P < 0.05), 2) increased sow feed consumption, 3) reduced sow weight loss during lactation (P < 0.05), 4) reduced sow weaning-estrus interval (P < 0.05), and 5) higher BW of piglets at weaning (P < 0.05). Additionally, a significant (P < 0.05) improvement in piglet birth weight and in the number of piglets weaned was observed in the second cycle of T2 sows, while a significant improvement of mean daily gain of piglets from birth to weaning was observed in the first cycle of T2 sows. Microbiological examination of fecal samples showed that probiotic treatment significantly reduced both Escherichia coli and Clostridium spp. in piglet feces, particularly during the second cycle. The data suggested that continuous feed supplementation with the probiotic is beneficial for both sows and piglets, since zootechnical benefits were observed in both cycles.

The commercial impact of pig Salmonella spp. infections in border-free markets during an economic recession.

The genus Salmonella, a group of important zoonotic pathogens, is having global economic and political importance. Its main political importance results from the pathogenicity of many of its serovars for man. Serovars Salmonella Enteritidis and Salmonella Typhimurium are currently the most frequently associated to foodborne infections, but they are not the only ones. Animal food products contaminated from subclinically infected animals are a risk to consumers. In border free markets, an example is the EU, these consumers at risk are international. This is why, economic competition could use the risk of consumer infection either to restrict or promote free border trade in animals and their products. Such use of public health threats increases during economic recessions in nations economically weak to effectively enforce surveillance. In free trade conditions, those unable to pay the costs of pathogen control are unable to effectively implement agreed regulations, centrally decided, but leaving their enforcement to individual states. Free trade of animal food products depends largely on the promotion of safety, included in "quality," when traders target foreign markets. They will overtake eventually the markets of those ineffectively implementing agreed safety regulations, if their offered prices are also attractive for recession hit consumers. Nations unable to effectively enforce safety regulations become disadvantaged partners unequally competing with producers of economically robust states when it comes to public health. Thus, surveillance and control of pathogens like Salmonella are not only quantitative. They are also political issues upon which states base national trade decisions. Hence, the quantitative calculation of costs incurring from surveillance and control of animal salmonelloses, should not only include the cost for public health protection, but also the long term international economic and political costs for an individual state. These qualitative and qualitative costs of man and animal Salmonella infections should be calculated in the light of free trade and open borders. Understandably, accurate calculation of the economic and political costs requires knowledge of the many factors influencing nationally the quality and safety of pork products and internationally free trade. Thus, how Salmonella pig infections affect commerce and public health across open borders depends on a state's ability to accurately calculate costs for the surveillance and control of animal salmonelloses in general, and pig infections as a particular example.

Effect of Bacillus licheniformis and Bacillus subtilis supplementation of ewe's feed on sheep milk production and young lamb mortality.

The purpose of this pilot study was to evaluate under field conditions the effect of a probiotic containing Bacillus licheniformis and Bacillus subtilis on young lamb mortality and sheep milk production when administered in the late pregnancy and lactation feed of ewes. In a sheep farm, two groups of milking ewes with identical genetic material, management, nutrition, health status and similar production characteristics were formed. One group (46 ewes) served as control, while the other one (48 ewes) served as a probiotic-treated group. Both groups of ewes received a similar feeding regiment, but the ewes of the second group were additionally offered a probiotic product containing B. licheniformis and B. subtilis (BioPlus 2B, Chr. Hansen, Denmark) at the approximate dose of 2.56 x 10(9) viable spores per ewe per day. Lamb mortality during the 1.5 months suckling period, and milk yield during the 2 months of milk collection for commercial purposes have been recorded. In the non-treated control group, 13.1% mortality was observed versus 7.8% in the probiotic-treated group (P = 0.33), with mortality being mainly due to diarrhoea. Microbiological examination of diarrhoeic faeces from some of the dead lambs in both groups revealed the presence of Escherichia coli. The average daily milk yield per ewe was significantly lower in the control group (0.80 l) than that in the probiotic-treated group (0.93 l) (P < 0.05). Fat and protein content of milk in ewes that received probiotics was significantly (P < 0.05) increased compared with untreated ewes. It was concluded that supplementing ewe's feed with probiotics may have beneficial effect on subsequent milk yields, fat and protein content.

Experimentally induced teat stenosis in dairy ewes: clinical, pathological and ultrasonographic features.

A strain of Staphylococcus chromogenes was introduced into the teat cistern of five ewes, teat inflammation and stenosis being the primary consequences. Initially, the inoculated teats were swollen and warm; later, a hard structure was palpated running lengthwise inside the teat, with a thick ring above the tip of the teat, which interfered with expression of milk. Mastitis, confirmed by clinical, cytological, bacteriological and histological findings, was evident 4 days after infection. Ultrasonographically, a hyperechoic line under the mucosa of the teat cistern was observed. At necropsy, the duct wall of the inoculated teats was found to be thickened. Histopathological features included leucocytic infiltration, especially under the mucosa of the teat, and extensive fibrosis in the subcutaneous tissues. S. chromogenes was recovered from scrapings from the duct and the cistern of the inoculated teats.

Prevalence of leptospira species among farmed and domestic animals in Greece.

A total of 1527 serum samples from pigs, goats, sheep, cattle and dogs in Greece were examined by the microscopic agglutination test and 11-8 per cent of them had antibodies against one or more Leptospira serovars at titres of 1/100 or more. The predominant serovar affecting farm animal species was Bratislava, and Copenhageni was common among dogs and the second most important serovar when all animals were considered together. Another prevalent serovar was Australis, but antibodies to Pomona were detected only in goats and cattle.

Antimicrobial resistance of animal and human strains of Enterobacteriaceae in Greece.

Two hundred sixty two strains of Enterobacteriaceae from animal and human sources where intermixing and/or spreading is possible were examined for their resistance to 15 antimicrobials frequently used in animal prophylaxis and metaphylaxis. The antimicrobials with the highest proportion of resistant strains from animal sources were amoxicillin 25 mg, colistin sulphate 25 mg, erythromycin 5 mg, penicillin G 10 mg and spectinomycin 10 mg. The same with isolates from human sources where ampicillin 10 mg, amoxicillin 25 mg, colistin sulphate 25 mg, erythromycin 5 mg, neomycin 10 mg, penicillin G 10 mg and spectinomycin 10 mg. Human isolates were resistant to more antimicrobials than animal isolates. Common a high proportion of isolates from both sources were resistant to some antimicrobials. Systematic and better distributed information is needed to address the epidemiological role of veterinary treatment, prophylaxis and metaphylaxis in antimicrobial resistance in Greece.

Serologic evidence of mixed infection involving the zoonoses leishmaniasis and leptospirosis in Greek dogs.

Leishmaniasis, an important zoonosis, was serologically found to coexist with leptospirosis, another important zoonosis. The proportion of dogs positive to leishmaniasis was approximately 36%. Significant differences were observed between dogs located in greater Athens and those from rural Greece. Although dogs from either of the groups had a similar chance to be infected, rural dogs had significantly (p<0.01) higher titers (1/1,600) than dogs from greater Athens. Thirty two of the 344 dog serum samples examined had positive antibody titers against Leptospira spp., but only 13 of them had titers of 1/400 or over to both of the infectious agents. Whilst noted differences in the antibody titers of samples with evidence of leishmaniasis and leptospirosis were not observed their coexistence may complicate the clinical outcome of cases of mixed infection.

Production and novel quantification of haemolysins produced by coagulase-negative staphylococci isolated from subclinical mastitis in sheep.

Coagulase-negative staphylococci producing cell-damaging toxins were isolated from the milk of sheep with subclinical mastitis. The haemolytic activity of Coagulase-negative staphylococcal strains was assessed on solid and liquid culture media. More than 61% and 76% of the tested strains on solid media produced evidence of alpha- and delta- haemolysins and more than 78% produced synergistic haemolysis. However almost all isolates producing haemolysin in liquid culture media produced only very few units of haemolysin compared to the positive control of five Coagulase-positive strains of staphylococci. It was concluded that solid media are better for classifying Coagulase-negative staphylococci as producers or not of haemolysins, and liquid media for measuring the size of this activity within the first few hours of intramammary infection.

A comparison of methods used in species identification of coagulase-negative staphylococci isolated from the milk of sheep.

Thirty reference strains of staphylococci, 76 strains isolated from confirmed cases of subclinical mastitis and 10 strains from the teat tip skin and the mouth of sucking lambs were assigned to species using four biochemical methods. These were the commercially available micromethods API Staph and Staph-Zym; the specialized laboratory method Rosco Set and Reactions in Standard Laboratory Culture Media (SLCM). The Rosco Set assigned species to all the strains, the SLCM to 96.5%, the Staph-zym to 86.1% and the API Staph to 81.4%. API Staph and the SLCM favoured the 'aureus' group with the 'epidermidis' second, while the reverse was true for the other two methods. All the identification methods assigned the same species to only 29% of the tested isolates, causing considerable variation in the number of species identified within each group. This variation indicates that reported species prevalence from cases of ovine mastitis should be viewed with caution.

Histopathological and ultrastructural observations of ovine mammary glands experimentally inoculated with coagulase-negative staphylococci.

The mammary glands of 21 primiparous Mule ewes were infected experimentally with one or other of seven isolates of coagulase-negative staphylococci belonging to one of four different species. All isolates caused inflammation that contributed to histopathological and ultrastructural changes. Histopathological changes varied from various degrees of neutrophilic inflammation at the early stages of infection to extensive mononuclear cell infiltration and development of fibrotic tissue at the late stages. The severity and extent of the lesions varied between isolates. At late stages of infection mononuclear cells were involved in active phagocytosis more often than neutrophils. Phagocytosed cocci within mononuclear cells were observed in the interstitium. These cells could become reservoirs of staphylococci that may prolong the inflammatory response.

In vivo presence of capsular polysaccharide in coagulase-negative staphylococci of ovine origin.

Three strains of coagulase-negative staphylococci identified as Staphilococcus warnery, Staphylococcus simulans and Staphylococcus haemolyticus were examine for evidence of capsular polysaccharide, in vitro by negative staining with India ink, and in vivo by transmission electron microscopy. In vitro, unstained materials surrounded clusters or single cocci. In vivo, capsula materials were surrounding phagocytosed bacteria cells. These capsular materials were either closely or loosely bound to bacterial cells, or projecting like "pili" from the surface of the cell wall. The evidence suggests that capsular polysaccharide is a common characteristic of coagulase-negative staphylococci causing prolonged intramammary infection of sheep.

Isolation of coagulase-negative staphylococci from the milk and environment of sheep.

Various species of coagulase-negative staphylococci (C-NS) are reported to be common in milk and on the teat skin of domestic ruminants. The commonest C-NS species in mastitic milk of cows varies between reports, with Staphylococcus simulans (Jarp, 1991) in one and Staph. hyicus in another (Watts & Washburn, 1991). The teat skin of heifers may be colonized by Staph. xylosus or Staph. chromogenes, while Staph. chromogenes and Staph. warneri are reported as frequent isolates from teat canals and secretion (Boddie & Nickerson, 1986; White et al. 1989). Staph. haemolyticus was isolated frequently from the nares, the teat skin and the milk of goats (Valle et al. 1991), although others reported Staph. xylosus (Bedidi-Madani et al. 1992) or Staph. epidermidis and Staph. capitis (Kalogridou-Vassiliadou, 1991) as the most predominant C-NS in goats' milk. Staph. simulans has been found experimentally to be pathogenic for the mammary gland of meat ewes (Fthenakis & Jones, 1990), but little is known about the prevalence of this species in ewes' milk collected from cases of naturally occurring subclinical mastitis (SCM). The aim of the present investigation was the identification of the commonest C-NS species in ewes' milk collected from field cases of SCM or predominating in the ewes' environment.

Electron microscopic study of milk sediments. Qualitative and quantitative observations.

Sediment of milk from ovine mammary glands infected experimentally with coagulase-negative staphylococci was examined by transmission electron microscopy. The proportions, of the various particles and cells present in milk and of macrophages and neutrophils involved in phagocytosis were determined 6 h, 18 h and 49 days after infection. All cell types together predominated over cytoplasmic bodies in approximately 54% of normal milk samples. The proportion of macrophages in normal milk was higher than that of neutrophils in approximately 69% of samples examined. After infection and in the early phase of inflammation, the proportion changed in favour of neutrophils. In both the early and late phases of infection, greater numbers of macrophages (3-27.2%) than neutrophils (0-2.7%) contained phagocytized cocci. Moreover, greater numbers of cocci were observed in macrophages than in neutrophils (mean values 8.1 vs 1.2), indicating the importance of the macrophage in the maintenance of ovine subclinical mastitis caused by coagulase negative staphylococci.

Leukotoxic factors produced by staphylococci of ovine origin.

Leukotoxins produced by staphylococci, especially Staphylococcus aureus, have long been considered important virulence determinants. The present assay examined 26 strains of staphylococci isolated from sheep for their ability to produce factors leukotoxic to polymorphonuclear leukocytes deriving from ovine mammary glands. Twenty one strains were coagulase-negative staphylococci and five coagulase-positive. Of the coagulase-negative staphylococci 17 were isolated from subclinical mastitis and four from the teat skin. The coagulase-positive strains were isolated from clinical mastitis and caused death to 91-100% of the cells. The coagulase-negative strains isolated from subclinical mastitis caused death to less than 50% of cells, while those from the skin did not affect any cells. The concentration of active leukotoxins is an important staphylococcal virulence factor. Low production of leukotoxins or no production by coagulase-negative staphylococci may determine their ability to survive the mammary gland defenses or cause clinical mastitis.

Isolation of Pasteurella haemolytica from grass, drinking water, and straw bedding used by sheep.

Pasteurella haemolytica was isolated from three of 18 grass samples and four of 18 water samples collected from two grazing fields occupied by sheep. This microorganism was also isolated from three of nine straw bedding samples collected from a pen housing ewes affected by mastitis caused by P. haemolytica. The same ewes developed scabbed papilloma-like lesions on the teat and udder skin. These lesions were colonized by P. haemolytica of various serotypes. Colder, wetter weather seems to prolong the survival of P. haemolytica in the environment of sheep. Survival of virulent strains of P. haemolytica in the environment could accumulatively increase the bacterial count, contributing to their transmission from animal to animal. The preference of P. haemolytica for colder, wetter conditions was confirmed in the laboratory where this microorganism survived longer in distilled water, phosphate-buffered saline, Todd-Hewitt broth, and ewe's milk kept at 4 degrees C.

Dynamics of intramammary infection in the sheep caused by coagulase-negative staphylococci and its influence on udder tissue and milk composition.

Over an entire lactation 891 milk samples were collected from 99 ewes in a well-managed dairy flock. Coagulase-negative staphylococci were isolated from 53 (5.9 per cent) of the samples, with 13 (6.6 per cent) of the glands giving coagulase-negative staphylococci in two or more consecutive samples. When a somatic cell count threshold of 6 x 10(5)/ml of milk was adopted as an indication of inflammation, 30 of the milk samples collected during early and mid-lactation, which contained coagulase-negative staphylococci, were considered to come from glands suffering subclinical mastitis. In five glands subclinical mastitis persisted for the entire lactation. A comparison of somatic cell counts measured with the fossomatic or Coulter counters suggested that the former gave the most reliable values, particularly in late lactation. After the experimental infection of 20 glands of meat breeds with coagulase-negative staphylococci, five glands remained infected for all 49 days of observation and seven glands excreted bacteria intermittently. Irrespective of the presence of coagulase-negative staphylococci in the sample, the composition of the milk from the challenged glands was significantly modified (P < 0.0001 for the infected, and P < 0.01 for the uninfected glands). Fat and protein concentrations were increased and lactose decreased, suggesting that although coagulase-negative staphylococci could not always be isolated, they persisted in many of the challenged glands and continued to influence the physiology of the gland. This possibility was supported by the presence of severe damage to the udder tissue of glands in which bacteria had been shown to be present for long periods.

Udder orf infection and its role in ovine clinical mastitis caused by Pasteurella haemolytica.

During an experimental study of ovine subclinical mastitis caused by coagulase-negative staphylococci, an outbreak of contagious ecthyma occurred among ewes unvaccinated against parapox virus. The same group of ewes developed a high rate (43.7%) of clinical mastitis caused by Pasteurella haemolytica. The rate of clinical mastitis among ewes vaccinated against parapox virus was very low (3.7%) suggesting that the presence of orf in the unvaccinated ewes was contributing to the high rate of clinical mastitis. An examination of the iron, sodium, potassium and albumin concentration of milk collected from 16 unvaccinated and nine randomly selected vaccinated ewes before experimental infection with coagulase-negative staphylococci or their uninfected control mammary glands indicated significant differences in the iron (p < 0.0001) and sodium (p = 0.01) concentration. Increased iron concentration in the milk may have assisted in the development of udder infection caused by P. haemolytica as iron is easily utilised by this bacterium.

Evaluation of the Staph-Zym system in the identification of staphylococci isolated from ovine milk and the ewe environment.

The Staph-Zym and the Rosco Set methods were used to assign species to 142 strains of staphylococci isolated from the milk of sheep with subclinical intramammary infection or teat skin, mouth of lambs or milking machine teat cups. One hundred eleven of the same strains were also tested by Standard Laboratory Culture Media. The Rosco Set assigned species to all strains tested, but the other two methods were unable to assign species to a similar proportion of isolates. Seventy six (68.5%) of the strains tested with all three methods were not assigned the same species, each method shown a preference for particular species. The Staph-Zym and the Rosco Set identified most of the strains as members of the "epidermidis" group with the "aureus" second, while the reverse was observed with the Standard Laboratory Culture Media. Only in the case of Staphylococcus epidermidis all methods were in close agreement. When the results of the Staph-Zym and the Rosco Set are compared, although they agree on the predominant group, they disagree on the predominant species within the same group.

Serum and milk iron levels during sheep intramammary infection caused by coagulase-negative staphylococci.

The concentration of iron (Fe) in the milk and serum of sheep was determined before and during experimental intramammary infection (IMI) by coagulase-negative staphylococci (C-NS). Fe concentration of normal milk and serum samples was 0.24 microgram/mL and 1.56 micrograms/mL respectively. Presence of C-NS in the mammary gland resulted in a significant increase in milk-iron concentration (p < 0.001) and a decrease in the serum-iron concentration. Serum-iron concentration was significantly decreased (p = 0.04) one d after the intramammary introduction of C-NS and 29 d later (p = 0.03).

Resistance of coagulase-negative staphylococci isolated from sheep to various antimicrobial agents.

Staphylococcal resistance to a variety of antimicrobial agents, defined as inhibition of bacteria growth in the presence of an antimicrobial agent, was approximately 75 per cent among coagulase-negative staphylococci isolated from milk and the teat skin of sheep. Resistance to broad spectrum antibiotics such as chloramphenicol and tetracycline as well as resistance to the combination of trimethoprim/sulphamethoxazole commonly used for the treatment of animals, was high in both groups of isolates. Four isolates of Staphylococcus epidermidis and three Staph xylosus isolated from the milk of dairy ewes were resistant to methicillin. Methicillin resistance is prevalent among human staphylococcal isolates and resistance to this antibiotic may reflect human handling of sheep.