Growth inhibitory effect of selected medicinal plants from Southern Ethiopia on the mycelial phase of Histoplasma capsulatum var. farciminosum.

BACKGROUND: Epizootic lymphangitis is an infectious and chronically debilitating disease of the equines. Histoplasma capsulatum var. farciminosum, a thermally dimorphic fungi, is the causative agent for the disease. In Ethiopia, the disease significantly affects carthorses, posing threats to animal welfare, and resulting in substantial economic losses. Limited availability of widely accessible antifungals in addition to the chronic nature of the disease is the major challenge against management of epizootic lymphangitis. This study aimed to assess the in vitro efficacy of specific local medicinal plant extracts against the mycelial phase development of H. capsulatum var. farciminosum in southern Ethiopia. The leaves of Xanthium strumarium, Kanda (Family Rubiaceae), Croton macrostachyus (Bisana in Amharic), and Centella Asiatica (Echere waye as a local name in Zeyissegna) that are traditionally used for the treatment of different skin ailments were collected and extracted for the in vitro trial. RESULTS: The study revealed that methanol extracts of Xanthium strumarium, Kanda, Croton macrostachyus, and Centella Asiatica, at minimum inhibitory concentrations of 1.25 mg/ml, 2.5 mg/ml, 2.5 mg/ml, and 5 mg/ml, respectively, inhibited the growth of H. capsulatum var. farciminosum. CONCLUSION: This in vitro finding could serve as significant preliminary data in the exploration of effective alternative treatment options for epizootic lymphangitis. This study provides a crucial foundation for further research aimed at determining the chemical components and in vivo effectiveness of these plant extracts against both the mycelial and yeast forms of Histoplasma capsulatum var. farciminosum.

Comparison of five basal compositions of selective chocolate agar media for isolation of Taylorella equigenitalis.

The gold standard method to isolate and identify Taylorella equigenitalis, the contagious agent of equine metritis, is the culture method according to the World Organisation for Animal Health Terrestrial Manual. No selective T. equigenitalis chocolate agar medium has been developed since the 1980s and the existing media show limited performances due to the fastidious nature of T. equigenitalis and the presence of interfering bacteria in the genital tract of equines. Here, the growth rates of 6 T. equigenitalis strains and 7 non-T. equigenitalis strains were compared on Timoney's selective medium formulated with 5 different basal agars (Columbia, Eugon, Blood, Mueller-Hinton and Tryptose Blood) provided by 2 to 4 suppliers per basal agar. The impact of glucose and/or Vitox supplementation was also investigated. Overall, the performance of selective T. equigenitalis media could be improved by substituting Eugon or Columbia agar with Blood, Mueller-Hinton or Tryptose Blood agar. It is nevertheless essential to validate the basal agar/supplier pair using a panel of T. equigenitalis strains. Furthermore, our findings confirm the need to supplement the selective media with a mixture of amino acids, nucleotides, and organic, mineral and vitamin compounds, translated here by Vitox supplementation.

Leptospiral antibody prevalence and surgical treatment outcome in horses with Equine Recurrent Uveitis (ERU) in Switzerland.

OBJECTIVE: To evaluate leptospiral antibody prevalence in 65 horses with ERU and compare outcome in 36 surgically treated eyes (2010-2015). PROCEDURES: Retrospective data analysis of horses with ERU (n = 65). C-value calculation with microagglutination assay titer (MAT) results for Leptospira spp. Evaluation of follow-up data after pars plana vitrectomy (PPV, n = 21 eyes) and suprachoroidal cyclosporine device implantation (SCDI, n = 15 eyes). Differences between groups were statistically analyzed using Fishers exact test, significance set at P < .05. RESULTS: Positive leptospiral titers were found in 28/65 blood, 31/65 aqueous humor (AH), and 19/20 vitreal (post-PPV) samples. The most common intraocular serovars were Leptospira interrogans grippotyphosa, pomona, and bratislava. Intraocular antibody production was suspected in samples of 22 horses (c-values > 1). Mean follow-up of surgical cases was 3.8 years (PPV) and 3.4 years (SCDI). PPV was performed in 21 eyes with positive, SCDI in 15 eyes with negative leptospiral test results. Uveitis recurred less often after PPV (2/21) compared to SCDI (6/15, P = .04). Retinal detachment occurred after PPV only (5/21, SCDI 0/15, P = .06), whereas only SCDI-treated eyes were enucleated (PPV 0/21, SCDI 3/15, P = .06). Blindness or visual impairment was equally likely to occur in both treatment groups after surgery (PPV 7/21, SCDI 7/15, P = .5). CONCLUSIONS: Leptospiral antibody prevalence is high in horses with ERU in Switzerland. Recurrence of uveitis is uncommon following PPV in the present study; an increased risk of retinal detachment exists. Enucleation is more often warranted in horses after SCDI in this study due to a higher uveitis recurrence.

The antimicrobial activity of bupivacaine, lidocaine and mepivacaine against equine pathogens: An investigation of 40 bacterial isolates.

Lameness is the most commonly reported health problem in horses, and lameness investigations which include local anaesthetic injections are routinely performed by equine practitioners. Through this process, bacteria can enter the tissues perforated by the needle and may cause local infections at the injection site. The objective of this in vitro study was to investigate if local anaesthetics at concentrations available in commercially available solutions could inhibit growth and/or kill bacteria that could be inoculated into the synovial space or soft tissues during injection. This study evaluated the antimicrobial activity of the local anaesthetics bupivacaine, lidocaine and mepivacaine against 40 equine clinical bacterial isolates of the Actinobacillus, Corynebacterium, Enterobacter, Escherichia, Pseudomonas, Rhodococcus, Staphylococcus and Streptococcus genera. Minimum inhibitory and minimum bactericidal concentrations (MICs and MBCs) were determined by the broth microdilution method. Clinically applied concentrations of bupivacaine, lidocaine, and mepivacaine inhibited visual growth of 93%, 93%, and 80% of isolates tested, respectively. For the majority (80%) of the inhibited isolates, the concentrations were also bactericidal. The tested local anaesthetics possessed antimicrobial activity against equine pathogens at concentrations that are routinely applied in clinical cases. However, this antimicrobial activity should not discourage antiseptic preparation prior to local anaesthetic injections.

Zoonotic necrotizing myositis caused by Streptococcus equi subsp. zooepidemicus in a farmer.

BACKGROUND: Streptococcus equi subsp. zooepidemicus is a beta-hemolytic group C streptococcus mainly causing infections in domesticated animals. Here we describe the first case of zoonotic necrotizing myositis caused by this bacterium. CASE PRESENTATION: The patient was a 73-year-old, previously healthy farmer with two asymptomatic Shetland ponies in his stable. After close contact with the ponies while feeding them, he rapidly developed erythema of his left thigh and sepsis with multiple organ failure. The clinical course was severe and complicated, requiring repetitive surgical excision of necrotic muscle, treatment with vasopressors, mechanical ventilation and continuous venovenous hemofiltration, along with adjunctive hyperbaric oxygen therapy. The patient was discharged from hospital at day 30, without obvious sequelae. The streptococcal isolate was identified as Streptococcus equi by MALDI-ToF MS, and was later assigned subspecies identification as S. equi subsp. zooepidemicus. Multilocus sequence typing identified the strain as a novel sequence type (ST 364), closely related to types previously identified in horses and cattle. A focused proteomic analysis revealed that the ST 364 expressed putative virulence factors similar to that of Streptococcus pyogenes, including homologues of the M protein, streptodornases, interleukin 8-protease and proteins involved in the biosynthesis of streptolysin S. CONCLUSION: This case illustrates the zoonotic potential of S. equi subsp. zooepidemicus and the importance of early clinical recognition, rapid and radical surgical therapy, appropriate antibiotics and adequate supportive measures when necrotizing soft tissue infection is suspected. The expression of Streptococcus pyogenes-like putative virulence determinants in ST 364 might partially explain the fulminant clinical picture.

Fighting Off Wound Pathogens in Horses with Honeybee Lactic Acid Bacteria.

In the global perspective of antibiotic resistance, it is urgent to find potent topical antibiotics for the use in human and animal infection. Healing of equine wounds, particularly in the limbs, is difficult due to hydrostatic factors and exposure to environmental contaminants, which can lead to heavy bio-burden/biofilm formation and sometimes to infection. Therefore, antibiotics are often prescribed. Recent studies have shown that honeybee-specific lactic acid bacteria (LAB), involved in honey production, and inhibit human wound pathogens. The aim of this pilot study was to investigate the effects on the healing of hard-to-heal equine wounds after treatment with these LAB symbionts viable in a heather honey formulation. For this, we included ten horses with wound duration of >1 year, investigated the wound microbiota, and treated wounds with the novel honeybee LAB formulation. We identified the microbiota using MALDI-TOF mass spectrometry and DNA sequencing. In addition, the antimicrobial properties of the honeybee LAB formulation were tested against all wound isolates in vitro. Our results indicate a diverse wound microbiota including fifty-three bacterial species that showed 90 % colonization by at least one species of Staphylococcus. Treatment with the formulation promoted wound healing in all cases already after the first application and the wounds were either completely healed (n = 3) in less than 20 days or healing was in progress. Furthermore, the honeybee LAB formulation inhibited all pathogens when tested in vitro. Consequently, this new treatment option presents as a powerful candidate for the topical treatment of hard-to-heal wounds in horses.

Development of ELISA test for determination of the level of antibodies against Rhodococcus equi in equine serum and colostrum.

Rhodococcus equi infection occurs worldwide and is one of the major causes of losing foals in the first six months of life. The application of serological tests in the diagnostics of rhodococcosis is limited, however they play a crucial role in immunological studies. The objective of this study was to develop and standardize ELISA test for the determination of the level of antibodies against Rhodococcus equi in equine serum and colostrum.Bacterial cell lysate was used as antigen. The test was standardized on 175 sera obtained from adult horses kept on rhodococcosis-free and endemic farms. Positive and negative control sera were used. The test detected IgG antibodies mainly against VapA protein, which was confirmed by Western blot analysis. The test was easy to perform, did not require inactivation of sera and had low well-to-well variation. The shelf life of antigen-coated ELISA plates was 21 days.The test allowed to reveal significant increase of R. equi-specific antibodies in both serum and colostrum in response to the vaccination (p<0.001). Therefore it can be applied to the evaluation of efficacy of immunization. Moreover, no statistically significant difference in the baseline antibody level in adult horses from rhodococcosis-free and endemic farm was revealed (α=0.05).

Changes in faecal bacteria and metabolic parameters in foals during the first six weeks of life.

Many foals develop diarrhoea within the first two weeks of life which has been suggested to coincide with postpartum oestrus in their dams. To analyse the pathogenesis of this diarrhoea we have determined faecal bacteria in foals and their dams (n=30 each), and serum IGF-1 and γ-globulins for 6 weeks after birth. In addition, effects of ß-carotene supplementation to mares (group 1: 1000 mg/day, n=15, group 2: control, n=15) on diarrhoea in foals were studied. Diarrhoea occurred in 92 and 79% of foals in groups 1 and 2, respectively, but was not correlated with oestrus in mares. Beta-carotene supplementation was without effect on foal diarrhoea. In mares, bacterial flora remained stable. The percentage of foals with cultures positive for E. coli was low at birth but increased within one day, the percentage positive for Enterococcus sp. was low for 10 days and for Streptococcus sp. and Staphylococcus sp. was low for 2-4 weeks. By 4 weeks of age, bacterial flora in foals resembled an adult pattern. Concentration of serum IGF-1 was low at birth (group 1: 149 ± 11, group 2: 166 ± 17ng/ml), increased after day 1 (day 7 group 1: 384 ± 30, group 2: 372 ± 36) but at no time differed between groups. Serum γ-globulin concentration in foals was low before colostrum intake and highest on day 1 (p<0.001 over time). In conclusion, neonatal diarrhoea in foals does not coincide with postpartum oestrus in their dams but with changes in intestinal bacteria and is not influenced by ß-carotene supplementation given to mares.

Equine histoplasmosis: treatment trial in cart horses in Central Ethiopia.

Therapeutic effects of Sodium Iodide (NaI), Potassium Iodide (KI), ground berries of "Endod" (Phytolacca dodecandra) and Penstrip were evaluated on 70 cases of equine hitoplasmosis (EH). Response to each treatment was assessed using clinical examination of the lesions. Statistically significant difference (P = 0.0036) in therapeutic effect was observed among the different remedies. Cases treated either with a combination of NaI and Penstrip (F = 6.34, P = 0.004) or "Endod" and Penstrip (F = 3.64, P = 0.031) demonstrated significant response. The difference in response to treatment between early and advanced cases of EH was statistically significant (t = 2.22, P = 0.0148).

Antibodies to Aqx toxin of Actinobacillus equuli in horses and foals.

Actinobacillus equuli is found in the normal oral flora of horses, but has been associated with several diseases, and particularly with the usually fatal septicaemia in neonatal foals which is thought to be associated with a failure of the passive transfer of immunoglobulins via the colostrum. The Aqx protein of A equuli, belonging to the RTX family of pore-forming toxins, is also cytotoxic to horse lymphocytes. The presence of antibodies to Aqx was investigated in sera from individual horses from different regions; the sera from adult horses and foals 24 hours after birth reacted with Aqx, and sera from foals sampled shortly after an intake of colostrum also reacted with Aqx, but sera from foals taken before an intake of colostrum did not react with Aqx.

Final classification of Bisgaard taxon 9 as Actinobacillus arthritidis sp. nov. and recognition of a novel genomospecies for equine strains of Actinobacillus lignieresii.

Phenotypic characterization of bacteria from diseased and healthy horses identified 18 isolates as Bisgaard taxon 9 and 11 isolates as Actinobacillus lignieresii. All strains of taxon 9 were alpha-galactosidase- and raffinose-positive and showed variable fermentation of (+)L-arabinose and (-)D-sorbitol. Strains of A. lignieresii were negative for these characteristics, with the exception of raffinose. Two strains from the (-)D-sorbitol-negative group of taxon 9 showed a 16S rRNA similarity of 99-6%, while 99.5% similarity was found between two strains of the (-)D-sorbitol-positive group. DNA-DNA hybridization between the two strains representing the (-)D-sorbitol-negative group showed 98% binding, and their closest relationship was to a strain of A. lignieresii (64%). The two strains of the (-)D-sorbitol-positive group showed 83% binding and were related to the (-)D-sorbitol-negative group at a 76% DNA binding level. Actinobacillus arthritidis sp. nov. is proposed for 12 strains of the (-)D-sorbitol-positive group. Actinobacillus genomospecies 2 is suggested for the six strains of the (-)D-sorbitol-negative group. Phenotypically, strains of A. arthritidis and Actinobacillus genomospecies 2 differ in (-)D-sorbitol fermentation and can be separated from Actinobacillus equuli by being trehalose-negative, while a positive reaction for alpha-galactosidase separates the taxa from A. lignieresii. The type strain of A. arthritidis, CCUG 24862T, was isolated from a joint of a horse. Three equine isolates of A. lignieresii that could not be separated from the type strain by means of phenotypic characteristics showed 98.6-100% 16S rRNA similarity, but only 96.4-96.7% similarity to the type strain. DNA-DNA hybridization between two strains of this group showed 92% binding but only 70% binding to the type strain of A. lignieresii. Consequently, these equine isolates of A. lignieresii represent a new genomospecies of Actinobacillus, suggested as genomospecies 1 because phenotypic characteristics are not presently available to separate it from the type strain of A. lignieresii.

Allisonella histaminiformans gen. nov., sp. nov. A novel bacterium that produces histamine, utilizes histidine as its sole energy source, and could play a role in bovine and equine laminitis.

When cattle and horses are fed large amounts of grain, histamine can accumulate in the gastrointestinal tract, and this accumulation can cause an acute inflammation of the hooves (laminitis). When ruminal fluid from dairy cattle fed grain supplements was serially diluted in anaerobic MRS medium containing histidine (50 mM), histamine was detected at dilutions as high as 10(-7). The histidine enrichments were then transferred successively in an anaerobic, carbonate-based medium (50 mM histidine) without glucose. The histamine producing bacteria could not be isolated from the rumens of cattle fed hay; however, histamine producing bacteria could be isolated the feces of cattle fed grain and the cecum of a horse. All of the histamine producing isolates had the same ovoid morphology. The cells stained Gram-negative and were resistant to the ionophore, monensin (25 microM). The doubling time was 110 min, and the yield was 1.5 mg cell protein per mmol histidine. The G+C content was 46.8%. Lysine was the only other amino acid used, but lysine did not allow growth if histidine was absent. Because carbohydrate and organic acid utilization was not detected, it appeared that the isolates used histidine decarboxylation as their sole mechanism of energy derivation. 16s rRNA gene sequencing indicated that the isolates were most closely related to low G+C Gram-positive bacteria (firmicutes), but similarities were < or = 94%. Because the most closely related bacteria (Dialister pneumonsintes, Megasphaera elsdenii and Selenomonas ruminantium) did not produce histamine from histidine, we propose that these histamine producing bacteria be assigned to a new genus, Allisonella, as Allisonella histaminiformans gen. nov., sp. nov. The type strain is MR2 (ATCC BAA610, DSM 15230).

Passive transfer of maternal immunoglobulin isotype antibodies against tetanus and influenza and their effect on the response of foals to vaccination.

Influenza and tetanus-specific antibodies of the IgG sub-isotypes are posively transferred to foals via colostrum and inhibit their response to inactivated influenza vaccines and tetanus toxoid. High titres of influenza antibodies of IgGa and IgGb subisotypes and tetanus antibodies of the IgGa, IgGb and IgG(T) subisotypes were detected in postsucking serum samples collected from foals born to mares that had received booster doses of multicomponent vaccines during the last 2 months of gestation. Thereafter, titres declined in an exponential manner but were still detectable in all foals at age 26 weeks, regardless of whether they had been vaccinated prior to age 26 weeks. Mean +/- s.e. half-life of decline of influenza IgGa antibodies (27.0 +/- 2.3 days) was significantly shorter than that of influenza IgGb antibodies (39.1 +/- 2.7 days; P<0.005). Tetanus IgGa and IgGb antibodies declined with half-lives of 28.8 +/- 3.0 and 34.8 +/- 5.1 days, respectively. Titres of tetanus IgG(T) antibodies were substantially higher than those of influenza IgG(T) antibodies in postsucking samples and remained so through age 26 weeks, declining with a half-life of approximately 35 days. Postsucking titres of tetanus and influenza antibodies of the IgA isotype were low and declined rapidly to undetectable levels. Yearlings showed significant increases in titre of influenza IgGa, IgGb and IgG(T) subisotype antibodies but no increase in influenza IgA antibodies in response to 2 doses of multicomponent vaccines containing tetanus toxoid and inactivated influenza A-1 and A-2 antigens. Yearlings also showed strong tetanus IgGa, IgGb and IgG(T) subisotype responses to one dose of vaccine and a substantial further rise in titre in response to administration of a second dose 3 weeks later, but failed to show an increase in titre of tetanus IgA antibodies. The influenza and tetanus IgGa, IgGb and IgG(T) subisotype responses of 6-month-old foals to vaccination followed the same pattern as those shown by yearlings but titres were generally lower. In contrast, 3-month-old foals failed to show increases in titre of either influenza or tetanus IgG subisotypes in response to 2 doses of vaccine and generally needed 1-3 additional booster doses of vaccine to achieve titres similar to those achieved by yearlings after 2 doses. Based on the finding that maternal antibodies exert a significant inhibitory effect on the response of foals to tetanus toxoid and inactivated influenza antigens, it is recommended that primary immunisation of foals born to vaccinated mares should not commence before age 6 months.

Opsonic capacity of foal serum for the two neonatal pathogens Escherichia coli and Actinobacillus equuli.

Two of the most commonly isolated foal pathogens are Escherichia coli and Actinobacillus equuli. The hypothesis tested in this study was that young foals carry a lower opsonic capacity for these bacteria compared to adult horses. A flow-cytometric method for the phagocytosis of these by equine neutrophils was established. The opsonic capacity of serum from healthy foals from birth to age 6 weeks was evaluated and related to the concentrations of IgGa and IgGb. Phagocytosis of yeast was used as a control. Serum was required for phagocytosis, with higher concentrations for E. coli than for A. equuli. Ingestion of colostrum led to a significantly higher serum opsonic capacity. After that, there was no consistent age-related trend for opsonic capacity for the different microbes. Foal serum showed similar or higher opsonisation of E. coli and A. equuli compared to serum from mature individuals. During the studied period, the predominance among IgG subisotypes switched from IgGb to IgGa. Although the overall correlation between concentrations of IgG subisotypes and serum opsonic capacity was poor, sera with IgGb levels below 1.9 mg/ml induced lower opsonisation of E. coli and yeast, but not of A. equuli. Complement activation was important for opsonisation of all tested microbes. The results of this study are significant to the understanding of a key immunological facet in the pathophysiology of equine neonatal septicaemia in clinical practice.

Experimental inoculation of foals and pigs with an enterotoxigenic E. coli isolated from a foal.

Hemolytic E. coli strain 807-13, O149:NM:K88(STb+, LT+), was isolated from the feces of a neonatal diarrheic foal. E. coli 807-13 was examined for adhesion to brush border membranes (BBM) from foals, adult horses and pigs, and its pathogenicity was assessed in neonatal foals and pigs. E. coli 807-13 did not adhere to equine BBM but adhered to pig BBM. It did not cause diarrhea nor did it colonize the intestinal epithelium of 3 colostrum-deprived and 3 suckled foals challenged at 24 h of age. Acute ulcerative gastritis and acute suppurative gastritis were observed in 2 colostrum-deprived challenged foals, and acute neutrophilic enteritis was observed in 1 colostrum-deprived and in 1 suckled challenged foal. No similar histopathologic lesions were detected in the control foals. Both gnotobiotic and suckled pigs developed diarrhea after challenge exposure to E. coli 807-13 and the intestinal epithelium of the pigs was colonized. Histopathologic evidence of gastritis and enteritis among the foals indicated some complicity of E. coli 807-13 in foal enteric disease.

Equine mastitis--a review of 28 cases.

Mastitis was diagnosed in 28 mares. Cytological evaluation of milk samples showed large numbers of neutrophils in 72 per cent and bacteria in 33 per cent. Aerobic bacteria were cultured from 71 per cent of samples. Streptococcus zooepidemicus was the most common isolate (37 per cent). Gram-negative species accounted for 42 per cent. Determination of the probability for antimicrobial susceptibility of bacterial isolates from mares with mastitis indicated that a trimethoprim-sulphonamide combination would be active against more than 75 per cent of isolates while penicillin would be active against less than 60 per cent. Systemic antibiotics, frequent milking, hotpacks and/or hydrotherapy and, if possible, infusion of an intramammary antibiotic preparation is recommended for treatment of equine mastitis.