In vitro bactericidal efficacy of equine polymorphonuclear leukocytes against Corynebacterium equi

Polymorphonuclear leukocytes from adult horses were separated from whole blood, using a 2-step Percoll gradient, and were tested for bactericidal function against Corynebacterium equi. Staphylococcus aureus, an organism against which equine neutrophils have proved efficacy, was a positive control. The percentage of uptake after a 15-minute preincubation of the neutrophils and bacteria in the presence of normal horse serum was also calculated. The results indicated that equine neutrophils effectively phagocytosed and killed C equi and S aureus. The percentage of uptake for S aureus (95% +/- 3%) was greater than that for C equi (85% +/- 6%) (P less than 0.001), but the bactericidal efficacy was equivalent. More than 90% of the ingested or attached bacteria were destroyed during the 3-hour incubation period (mean percentage of C equi killed = 96 +/- 2%; mean percentage of S aureus killed = 91 +/- 8%). These results indicated that a failure of bacterial killing by neutrophils is unlikely to be important in the pathogenesis of C equi pneumonia in the horse.     

Lymphocyte stimulation response in horses against phytohaemagglutinin and M protein of Streptococcus equi using whole blood.

Lymphocyte stimulation was observed in whole equine blood in the presence of phytohaemagglutinin and M protein extracted from a typical strain of Streptococcus equi. Blood samples were collected from several healthy horses and horse and pony foals and cultured in vitro with varying concentrations of phytohaemagglutinin and M protein for several days. Phytohaemagglutinin was found to induce lymphocyte stimulation in these animals. Highest mean stimulation indices in horse foals (49.3 +/- 24.4) and pony foals (54.7 +/- 32.0) were observed with 0.625 and 1.25 micrograms/mL phytohaemagglutinin, respectively, at either 72 or 96 hours of incubation. Significantly higher radioactive counts per minute in horse and pony foals were recorded in blood cultures incubated with 0.625 and 1.25 micrograms/mL phytohaemagglutinin. M protein induced a dose related stimulation response in adult horses. Maximum stimulation indices were observed against 125 micrograms/mL M protein at 96 hours. These stimulation indices were higher in adult horses (40.0 +/- 2.2) than observed in pony foals (14.4 +/- 15.7). Higher stimulation levels in adult horses indicated either nonspecific stimulation against M protein or previous exposure of these animals to S. equi.     

The interaction of Rhodococcus equi and foal neutrophils in vitro

Polymorphonuclear neutrophil leukocytes (PMNL) from 8 healthy foals (2-14 weeks of age) and 2 foals with bacterial pneumonia were separated from whole blood using a 2 step Percoll gradient. Purified PMNL were tested for bactericidal function against Rhodococcus equi and Staphylococcus aureus in the presence of normal horse serum. The percentage uptake after a 15-min pre-incubation of PMNL and bacteria was also calculated. Ultrastructural examination of the interaction of R. equi and normal foal PMNL was performed after 15 min incubation. Results indicated that foal PMNL effectively phagocytose and destroy R. equi and S. aureus in the presence of normal horse serum. The mean percent uptake for R. equi was 99.3 +/- 0.4% and for S. aureus 99.9 +/- 0.1%. Further, 97.8 +/- 0.1% ingested R. equi and 98.4 +/- 0.1% ingested S. aureus were destroyed in the 15-min incubation period. Over the 3-h incubation, 91.9% of remaining R. equi were killed, but only 49.2 +/- 31.9% of S. aureus (P less than 0.01). Total bactericidal effect of foal PMNL, however, was 99.3 +/- 0.4% against R. equi and 99.9 +/- 0.1% against S. aureus. The percentage uptake and total bactericidal efficacy of neutrophils from sick foals was greater than 95%. Ultrastructural examination of the PMNL-R. equi interaction after 15 min incubation revealed phagocytosis of the bacteria and morphologic changes consistent with neutrophil degranulation. This study suggests that a defect in PMNL bactericidal capability is not likely to be a contributing factor in the pathogenesis of R. equi pneumonia in foals.     

Selection of an aminoglycoside antibiotic for administration to horses

The serum concentrations of the aminoglycosides neomycin, kanamycin and streptomycin were determined after intravenous (iv) and intramuscular (im) administration. These values were then related to the minimum inhibitory concentrations (MIC) of a number of equine pathogenic bacteria to determine the duration of therapeutic serum concentrations of the aminoglycosides in the horse. Pharmacokinetic analysis of the data using neomycin as the example revealed a mean (+/- sd) peak serum concentration of 23.2 +/- 10.2 micrograms/ml present at 30 mins, and at 8 h the serum concentration was 2.8 +/- 0.8 micrograms/ml. From the pharmacological analysis of concentration-time data it was shown that neomycin was very rapidly absorbed from the im injection site, with an absorption half-time of 0.16 +/- 0.05 and was well absorbed (systemic availability was 73.7 +/- 26.9 per cent). A peak tissue level, which represented 40 per cent of the amount of drug in the body, was obtained at 32 mins after injection of the drug. At 8 h, the fractions of the dose in the central and peripheral compartments of the model were 1.5 per cent and 2.5 per cent respectively, and 96 per cent was the cumulative amount eliminated up to that time. Based on the MIC values of the majority of isolates of Corynebacterium equi, and only a few isolates of Klebsiella pneumoniae, Escherichia coli, Salmonella typhimurium and Streptococcus equi, one would expect a serum concentration of more than 2 micrograms neomycin/ml up to 8 h following im dosage (10 mg/kg) to be therapeutically effective.     

Tracing outbreaks of Streptococcus equi infection (strangles) in horses using sequence variation in the seM gene and pulsed-field gel electrophoresis

Strangles is a serious respiratory disease in horses caused by Streptococcus equi subspecies equi (S. equi). Transmission of the disease occurs by direct contact with an infected horse or contaminated equipment. Genetically, S. equi strains are highly homogenous and differentiation of strains has proven difficult. However, the S. equi M-protein SeM contains a variable N-terminal region and has been proposed as a target gene to distinguish between different strains of S. equi and determine the source of an outbreak. In this study, strains of S. equi (n=60) from 32 strangles outbreaks in Sweden during 1998-2003 and 2008-2009 were genetically characterized by sequencing the SeM protein gene (seM), and by pulsed-field gel electrophoresis (PFGE). Swedish strains belonged to 10 different seM types, of which five have not previously been described. Most were identical or highly similar to allele types from strangles outbreaks in the UK. Outbreaks in 2008/2009 sharing the same seM type were associated by geographic location and/or type of usage of the horses (racing stables). Sequencing of the seM gene generally agreed with pulsed-field gel electrophoresis profiles. Our data suggest that seM sequencing as a epidemiological tool is supported by the agreement between seM and PFGE and that sequencing of the SeM protein gene is more sensitive than PFGE in discriminating strains of S. equi.     

Surveillance programme for important equine infectious respiratory pathogens in the USA

The prevalence and epidemiology of important viral (equine influenza virus [EIV], equine herpesvirus type 1 [EHV-1] and EHV-4) and bacterial (Streptococcus equi subspecies equi) respiratory pathogens shed by horses presented to equine veterinarians with upper respiratory tract signs and/or acute febrile neurological disease were studied. Veterinarians from throughout the USA were enrolled in a surveillance programme and were asked to collect blood and nasal secretions from equine cases with acute infectious upper respiratory tract disease and/or acute onset of neurological disease. A questionnaire was used to collect information pertaining to each case and its clinical signs. Samples were tested by real-time PCR for the presence of EHV-1, EHV-4, EIV and S equi subspecies equi. A total of 761 horses, mules and donkeys were enrolled in the surveillance programme over a 24-month study period. In total, 201 (26.4 per cent) index cases tested PCR-positive for one or more of the four pathogens. The highest detection rate was for EHV-4 (82 cases), followed by EIV (60 cases), S equi subspecies equi (49 cases) and EHV-1 (23 cases). There were 15 horses with double infections and one horse with a triple infection. The detection rate by PCR for the different pathogens varied with season and with the age, breed, sex and use of the animal.     

Streptococcus equi but not Streptococcus zooepidemicus produces potent mitogenic responses from equine peripheral blood mononuclear cells

Streptococcus equi causes equine strangles. The acute disease has many of the hallmarks of an acute response including high fever, elevated plasma fibrinogen and neutrophilia, affects known to be mediated by proinflammatory cytokines. The objective of this study was to screen-culture supernatants from equine clinical isolates of S. equi and S. zooepidemicus for stimulation of mitogenic responses by horse peripheral blood mononuclear cells. Mitogenicity comparable to that of concanavalin A was detected in culture supernatants of S. equi strains but not in those of S. zooepidemicus. Mitogenicity was neutralised by Proteinase K and a post-strangles convalescent serum, and evidence for the presence of both thermo-resistant and thermo-labile mitogenic factors was obtained. Release of proteinaceous immunogenic mitogens in combination with the antiphagocytic protein SeM unique to S. equi may therefore contribute to some of the severe clinical manifestations of acute strangles in the horse.     

Studies on the immunogenicity of Streptococcus equi vaccines in foals.

The ability of either formalin-treated or heat-inactivated whole Streptococcus equi cell vaccines or partially purified M-protein of S. equi to give rise to protective antibody levels was studied in Standardbred foals by serological means. Two commercial preparations, i.e. a beta-propiolactone killed whole S. equi cell bacterin and a cell-free extract of S. equi cells were included in the study. The mean passive hemagglutination antibody titers (10 X log2) in sera of foals given either four doses of formalin-treated whole cell vaccine or an initial dose of formalin-treated followed by three doses of heat-inactivated vaccine with or without levamisole were significantly higher two weeks after the final dose. These passive hemagglutination antibody titers were higher in foals given formalin-treated whole cell vaccine (6.7 +/- 1.5) than given commercial bacterin (4.5 +/- 2.1). The passive hemagglutination antibody titers in all the groups decreased at 12 to 16 weeks after fourth dose of the vaccine. Foals given a commercial cell-free extract did not show a significant increase in passive hemagglutination antibody titers even up to four weeks after third dose. A group of six pony foals immunized with partially-purified M protein showed mean passive hemagglutination antibody titers lower than those observed in foals given whole cell vaccines. In a challenge experiment with S. equi, two of six foals vaccinated with partially-purified M-protein and all three controls developed clinical disease. The passive hemagglutination antibody of vaccinated foals increased after challenge, while at 28 days postchallenge the passive hemagglutination antibody titers of vaccinates and recovered controls were similar.     

A comparison of different methods of extraction of the M-protein from Streptococcus equi.

The molecular weights of the proteins produced in different extracts of Streptococcus equi were compared on immunoblots with antisera against acid extracted and mutanolysin extracted M-protein. Acid and alkaline extracts of S. equi contained some peptides of similar molecular weight that reacted with antiserum against an acid extracted 41,000 m.w. fragment suggesting that these fragments contained common epitopes. Comparison of the amino acid compositions of the 35,000 m.w. fragment of the alkaline extract and the 41,000 m.w. fragment of the acid extract suggest that these immunologically reactive fragments were probably derived from the same protein. Little cross-reactivity was observed between antisera against S. equi acid extracted protein and the native 58,000 m.w. M-protein. This suggests that conformational epitopes on the native M molecule are not present after acid treatment.     

马腺疫链球菌fneB基因LAMP检测方法的建立及应用

为了建立快速简便的fneB-LAMP检测法用于马腺疫病原马链球菌马亚种(S.equi)的检测,本试验采用恒温水浴进行LAMP扩增,建立可视化fneB-LAMP检测方法.结果显示:对大肠埃希菌、无乳链球菌、金黄色葡萄球、蜡状芽孢杆菌和克雷伯杆菌5种菌与标准S.equi菌株检测,只能够检测出标准阳性菌株;对新疆昭苏县3个马...     

Se18.9, an anti-phagocytic factor H binding protein of Streptococcus equi

Evasion of phagocytosis is an important virulence determinant of Streptococcus equi (S. equi subsp. equi), the cause of equine strangles and distinguishes it from the closely related but much less virulent S. zooepidemicus (S. equi subsp. zooepidemicus). We describe Se18.9, a novel H factor binding protein secreted by S. equi but not by S. zooepidemicus that reduces deposition of C3 on the bacterial surface and significantly reduces the bactericidal activity of equine neutrophils suspended in normal serum for both S. equi and S. zooepidemicus. Se18.9 is secreted abundantly by actively dividing cells and is also bound to the bacterial surface. Strong serum and mucosal antibody responses are elicited in S. equi infected horses. Although a gene identical to se18.9 was not detected in S. zooepidemicus, sequences encoding proteins of similar size with similar signal peptide sequences were found in 3 of 12 randomly selected strains. Since Se18.9 is unique to S. equi, and immunoreactive with convalescent sera and mucosal IgA, it has potential for immunodiagnosis and for study of mucosal antibody response to S. equi.     

Effects of misoprostol and omeprazole on basal gastric pH and free acid content in horses

The basal gastric pH and free acid contents from five young adult healthy horses were determined at one hour intervals for eight hours. The basal gastric pH and free acid contents varied from 1.63 +/- 0.06 to 1.97 +/- 0.11 and 26.42 +/- 4.14 to 17.92 +/- 5.28 mmol litre-1, respectively. Misoprostol, a methylester analogue of prostaglandin (5 micrograms kg-1, orally) produced a time-dependent increase in the basal gastric pH to above 3.5 (P less than 0.05) at three, four and five hours after administration with a concomitant reduction of 80 to 90 per cent in the basal gastric free acid contents throughout the eight hour period monitored. Omeprazole, a benzimidazole derivative (0.5 mg kg-1, intravenously) increased the basal gastric pH to above 3.5 at two and three hours after administration with a concomitant reduction of 65 to 90 per cent in the basal gastric free acid contents for seven of the eight hour periods monitored. These results confirm that the horse is a basal acid secretor, and both misoprostol and omeprazole are effective inhibitors of the basal gastric acid secretion, thus establishing that both prostaglandins and H+/K+-ATPase play an important role in controlling parietal cell function of the equine gastric mucosa.     

The pathogenic equine streptococci

Streptococci pathogenic for the horse include S. equi (S. equi subsp. equi), S. zooepidemicus (S. equi subsp. zooepidemicus), S. dysgalactiae subsp. equisimilis and S. pneumoniae capsule Type III. S. equi is a clonal descendent or biovar of an ancestral S. zooepidemicus strain with which it shares greater than 98% DNA homology and therefore expresses many of the same proteins and virulence factors. Rapid progress has been made in identification of virulence factors and proteins uniquely expressed by S. equi. Most of these are expressed either on the bacterial surface or are secreted. Notable examples include the antiphagocytic SeM and the secreted pyrogenic superantigens SePE-I and H. The genomic DNA sequence of S. equi will greatly accelerate identification and characterization of additional virulence factors and vaccine targets. Although it is the most frequently isolated opportunist pyogen of the horse, S. zooepidemicus has been the subject of few contemporary research studies. Variation in the protectively immunogenic SzP proteins has, however, been well characterized. Given its opportunist behavior, studies are urgently needed on regulation of virulence factors such as capsule and proteases. Likewise, information is also very limited on virulence factors and associated gene regulation of S. dysgalactiae subspecies equisimilis. It has recently been shown that equine isolates of Streptococcus pneumoniae are clonal, a feature shared with S. equi. All equine isolates express capsule Type III, are genetically similar, and have deletions in the genes for autolysin and pneumolysin. In summary, the evolving picture of the interaction of the equine pathogenic streptococci and their host is that of multiple virulence factors active at different stages of pathogenesis. The inherent complexity of this interaction suggests that discovery of effective combinations of immunogens from potential targets identified in genomic sequence will be laborious.     

Acute recumbency associated with Anaplasma phagocytophilum infection in a horse

An 11-year-old Hanoverian-cross gelding was evaluated because of acute onset of ataxia, recumbency, and fever. At the stable, this and other horses had recently been infested with ticks. Results of analysis of a sample of CSF were within reference limits, but hematologic abnormalities included lymphopenia, thrombocytopenia, mild anemia, and intracytoplasmic inclusion bodies in neutrophils that were consistent with Anaplasma phagocytophilum (previously Ehrlichia equi). Results of serum biochemical analyses were characteristic of infection and included high, unconjugated bilirubin concentration. Other common causes of recumbency in horses, such as equine protozoal myeloencephalitis, infection with eastern or western equine encephalitis viruses and equine herpesvirus-1, West Nile viral encephalitis, trauma, and metabolic disease, were ruled out. The horse responded quickly to i.v. administration of oxytetracycline and recovered fully within 6 days.     

In vitro phagocytosis and killing of Corynebacterium equi by alveolar macrophages of foals

Bronchoalveolar lavage was performed 5 times, sequentially, on 3 healthy foals while each foal was 6 to 63 days of age. Phagocytosis and bactericidal assays were performed on recovered alveolar macrophages. Corynebacterium equi and alveolar macrophages at a ratio of 10:1 were incubated for 1 hour in medium containing 1% heat-inactivated rabbit anti-C equi serum. After incubation, greater than 90% of the alveolar macrophages contained at least 1 ingested bacterium and each alveolar macrophage contained 9.4 +/- 1.0 bacteria (mean +/- SE). After alveolar macrophages and C equi were incubated for 1 hour in medium containing heat-inactivated pooled normal horse serum, approximately 24% of the alveolar macrophages contained at least 1 bacterium and each alveolar macrophage contained 0.8 +/- 0.7 bacteria. From 6 to 61 days of age, each foal had significantly (P less than 0.05) decreased phagocytic activity by alveolar macrophages, but a significant change in killing of C equi by alveolar macrophages was not found in the foals from 21 to 61 days of age. After incubating alveolar macrophages and C equi for 4 hours in vitro, approximately 75% of ingested C equi remained viable.     

Selective medium for isolation of Haemophilus somnus from cattle and sheep

Incorporation of vancomycin (5 micrograms/ml), neomycin (5 micrograms/ml), sodium azide (50 micrograms/ml), nystatin (100 iu/ml) and cyclohexamide (100 micrograms/ml) into 5 per cent horse blood agar results in a selective medium for the primary isolation of Haemophilus somnus from cattle and sheep. Addition of thiamine monophosphate (1 microgram/ml) to the medium enhanced growth of this bacterium. Gram-positive bacteria did not grow on the medium and colonies of many Gram-negative bacteria were eliminated or reduced in numbers and size. Colonies of H somnus were larger on the selective medium than on sheep blood agar but retained typical morphology. Recovery of 18 laboratory strains was 73 to 166 per cent (mean 112) on selective medium compared to sheep blood agar. H somnus was isolated from the vagina of a total of 136 (28.6 per cent) of 476 cows surveyed, 79 (16.6 per cent) on sheep blood agar and 129 (27.1 per cent) on selective medium. The selective agents and thiamine were stable indefinitely as a freeze dried mixture while prepared plates were stable for two weeks.     

Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus

Strangles is a contagious equine disease caused by Streptococcus equi subsp. equi. In this study, clinical strains of S. equi (n=24) and Streptococcus equi subsp. zooepidemicus (n=24) were genetically characterized by sequencing of the 16S rRNA and sodA genes in order to devise a real-time PCR system that can detect S. equi and S. zooepidemicus and distinguish between them. Sequencing demonstrated that all S. equi strains had the same 16S rRNA sequence, whereas S. zooepidemicus strains could be divided into subgroups. One of these (n=12 strains) had 16S rRNA sequences almost identical with the S. equi strains. Interestingly, four of the strains biochemically identified as S. zooepidemicus were found by sequencing of the 16S rRNA gene to have a sequence homologous with Streptococcus equi subsp. ruminatorum. However, they did not have the colony appearance or the biochemical characteristics of the type strain of S. ruminatorum. Classification of S. ruminatorum may thus not be determined solely by 16S rRNA sequencing. Sequencing of the sodA gene demonstrated that all S. equi strains had an identical sequence. For the S. zooepidemicus strains minor differences were found between the sodA sequences. The developed real-time PCR, based on the sodA and seeI genes was compared with conventional culturing on 103 cultured samples from horses with suspected strangles or other upper respiratory disease. The real-time PCR system was found to be more sensitive than conventional cultivation as two additional field isolates of S. equi and four of S. zooepidemicus were detected.     

THE IDENTIFICATION OF BABESIA EQUI IN AUSTRALIA

A Babesia parasite, isolated from the blood of a horse at Bowral, New South Wales, was identified on the basis of its morphological features, host specificity and serological reactions, as Babesia equi (Laveran 1901). The case was originally reported by Churchill and Best (1976, Aust. vet. J. 52: 487) and is the first record of equine babesiosis in Australia. In preliminary studies, the organism produced only a mild disease in an intact horse, but caused the typical clinical syndrome of acute babesiosis in a splenectomised horse, which died 19 days after the intravenous inoculation of the parasites.     

IdeE reduces the bactericidal activity of equine neutrophils for Streptococcus equi

Streptococcus equi (S. equi) causes equine strangles, a highly contagious and widespread purulent lymphadenitis of the head and neck. Highly resistant to phagocytosis, it produces long extracellular chains in affected lymph nodes. In a screen of clones reactive with convalescent serum from a gene library of S. equi CF32 we identified IdeE, an IgG-endopeptidase and homologue of the leucocyte receptor Mac-1 (CD11b). IdeE is expressed during S. equi infection eliciting both serum and mucosal antibody responses which persisted at significant levels in serum for over 200 days. Release from S. equi into culture medium was detected during the exponential phase of growth. The closely related Streptococcus zooepidemicus appeared to store the protein but not to release it. Antiphagocytic activity for equine neutrophils was dose-dependent and neutralized by IdeE-specific antiserum. Biotinylated IdeE bound weakly to about 77% of purified equine neutrophils and strongly to the remainder.     

Demographic characteristics of the equine population of northern Britain.

The size, composition and distribution of the equine population of Scotland and the five northernmost counties in England were estimated through a series of mailed questionnaire surveys of sentinel veterinary practices and horse owners. An estimated 96,622 equine animals were kept by an estimated 26,114 owners. The mean (sd) age of the population was 11.0 (7.5) years (range one month to 37 years). Thoroughbred or thoroughbred-cross animals were the most numerous, constituting 30 per cent (95 per cent confidence interval 27 to 33 per cent) of the total population. The ratio of males:females was 1:1.