Genotypic characterization of VapA positive Rhodococcus equi in foals with pulmonary affection and their soil environment on a warmblood horse breeding farm in Germany

Pulsotypes of VapA positive Rhodococcus equi isolated from foals and soil on a farm in Germany were characterized on the basis of nasal and tracheal samples simultaneously collected in 2003 from 217 foals with sonographic evidence of pneumonia or pulmonary abscesses. Of the 217 double samples, R. equi was isolated in 118 (54%) of the tracheal samples and in 52 of the nasal swab samples (24%) (P < 0.001). Furthermore, 37 and 55 isolates were also randomly selected from nasal swabs and the tracheal samples, respectively, and further processed to determine the presence of VapA by colony blot enzyme-linked immunosorbent assay. This method showed that 26 (68%) of the nasal swabs and 40 (73%) of the tracheal samples were VapA-positive.R. equi was isolated from 56 (87%) of the 64 soil samples taken from the paddocks and stables in March and from 17 (68%) of the 25 samples taken in July of 2003. Three (21%) of these randomly selected 14 isolates from March and 13 (81%) of the 16 from July were VapA-positive.The VapA positive isolates from foals and soil were genotyped by plasmid profiling, restriction fragment length polymorphism analysis and pulsed-field gel electrophoresis. Of the 83 isolates, 80 contained an 85-kb type I plasmid and three contained an 87-kb type I plasmid. Pulsed-field gel electrophoresis yielded four distinct VspI profiles dividing 83 isolates into three major (A1, 51; D, 14; and 11 isolates) and three minor (C, 4; A3, 2; and A2, 1 isolates) groups. These results suggest that the majority of foals were exposed to and infected with three pulsotypes of VapA positive R. equi containing an 85-kb type I plasmid.     

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil

The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15–17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil.     

Resistance studies of erythromycin and rifampin for Rhodococcus equi over a 10-year period

This study sought to determine whether an increase in resistance of Rhodococcus equi to the antibiotics rifampin and erythromycin occurred over a 10-year period. This was carried out by the use of E test strips for rifampin and erythromycin to determine the MIC (minimum inhibitory concentration) values of Rhodococcus equi to this combination of antibiotics.The findings of this study indicated that there was an increase in resistance of Rhodococcus equi to rifampin and erythromycin over the 10-year period. The MIC for rifampin increased from 0.081 μg/ml in 1996 to 0.187 μg/ml in 2006 and from 0.258 μg/ml for erythromycin during the years prior to 2000 to 0.583 μg/ml in 2006.This finding suggests that there may be a problem in the treatment of Rhodococcus equi infections in foals in the future, particularly as the number of drugs available for treatment of Rhodococcus equi infection is limited because of the intracellular capabilities of this bacterium. Antibiotics used in its treatment have to be able to penetrate the polysaccharide cell wall of Rhodococcus equi as well as the alveolar macrophages in which the bacterium is capable of surviving.     

The relationship of soil province to molecular characterization and phylogenetic analysis of Cryptosporidium parvum isolated from calves in Georgia

Although Cryptosporidium spp. are found throughout the world and in multiple environmental conditions, few data are available that explore the possibility of an association between specific environmental parameters and the species or strain of Cryptosporidium. This study examines the potential association between a particular Cryptosporidium species/strain found in calves and soil provinces in Georgia, USA. Necropsy cases spanning the years 1996-2002 were tested. No significant differences (P=0.962, chi(2) test of homogeneity) between numbers of positive cases were noted among soil provinces. Phylogenetic analysis of the sequences for the PCR products revealed sequence similarity of the products with Cryptosporidium parvum strain C1. Although, clinical Cryptosporidiosis in calves was not found to be affected by soil province and may be caused by a single genotype, other genotypes may be responsible for subclinical infection and warrant further investigation.     

Effects of inactivated parapoxvirus ovis on the cumulative incidence of pneumonia and cytokine secretion in foals on a farm with endemic infections caused by Rhodococcus equi

The objectives of the present study were to determine if administration of inactivated parapoxvirus ovis (IPPVO) can decrease the cumulative incidence of pneumonia and increase the number of IFN-γ- and IL-4-secreting cells among foals. Fifty-nine foals were randomly assigned to 2 treatment groups (IPPVO or placebo) prior to birth. At 24-48 h of age, foals received 2 ml of either IPPVO or a placebo by intramuscular injection. Injections were repeated 24h and 8 days later. The number of IFN-γ- and IL-4-secreting cells was measured using a validated ELISPOT assay on blood mononuclear cells collected when the foals were 1-14 days old. Foals were monitored daily for clinical signs of pneumonia and biweekly for lung lesions by ultrasonography. The proportion of foals that developed clinical or ultrasonographic evidence of pneumonia was not significantly different between IPPVO (16 of 28) and placebo (14 of 31). IFN-γ- and IL-4-secreting cells were detected in only 22 and 15 foals, respectively. There was a significant effect of treatment with IPPVO on the number of IFN-γ secreting cells in foals 7- to 14-days-old but not in younger foals. There was no significant effect of treatment with IPPVO on the number of IL-4-secreting cells. The odds of detecting IFN-γ (5.1; 95% CI: 1.5-15) and IL-4 (3.5; 95% CI: 1.1-12) were significantly higher in foals 7-14 days than in younger foals regardless of treatment group. There was no significant association between IFN-γ or IL-4 secretion early in life and subsequent development of pneumonia.     

Parascaris equorum in foals and in their environment on a Swedish stud farm, with notes on treatment failure of ivermectin

Environmental contamination and the egg excretion pattern of the ascarid Parascaris equorum (Nematoda) was investigated in relation to anthelmintic treatment on a Swedish stud farm. Faecal samples from 15 foals, dewormed every 8th-week with a paste formulation of ivermectin at the standard dose rate of 0.2 mg/kg bodyweight, were collected at five sampling occasions between August and November 2006. In addition, soil samples were obtained from four paddocks used by these foals in November 2006. The number of eggs per gram (epg) was counted in both faeces and soil. Egg excretion started when the foals were 3-4 months, and reached the highest levels when they were approximately 5-month-old, and was then followed by a decline. Egg excretion seemed to be unaffected by ivermectin despite these foals were dewormed at regular intervals. In four out of five foals examined 10 days after treatment, epg actually increased. In contrast, when either fenbendazol or pyrantel embonate were used instead of ivermectin, treatments were effective. The number of eggs in soil was significantly higher in the permanent paddock compared to in the temporarily used soil paddock and in the summer paddocks.     

Preliminary findings of Salmonella spp. in captive green iguanas (Iguana iguana) and their environment

Captive reptiles are routinely identified as reservoirs of Salmonella spp. and reports of reptile-associated salmonellosis are increasing. Unfortunately, little is known about the epidemiology of Salmonella spp. and green iguanas. We did a limited survey of a green-iguana farm in El Salvador to identify sources of Salmonella spp. in green iguanas and their environment. A limited number of samples for microbiological culture were collected from iguanas (adult, hatchling, and embryos) and their environment (food, water, soil, shelter, insects, and wild-caught lizards). Salmonella spp. was isolated from the intestine of both adult (3/20) and hatchling iguanas (8/20). There was no evidence of Salmonella spp. in the reproductive tracts of female iguanas (0/10). Salmonella spp. was isolated from the surface of 40% (7/16) of the egg surfaces tested. Salmonella spp. was not identified from the externalized yolk-sac of the iguana embryos tested. Soil samples from a breeding pen and a nest were both positive for Salmonella spp. Eight different Salmonella spp. serotypes were identified in this survey. These results suggest that horizontal transmission of Salmonella spp. is a potential source of exposure to hatchling iguanas at this facility.     

Prevalence and age-related infection of Cryptosporidium suis, C. muris and Cryptosporidium pig genotype II in pigs on a farm complex in the Czech Republic

A total of 413 pig faecal samples were collected from pre-weaners (119), starters (131), pre-growers (123) and sows (40) from a farm with a closed breeding system segmented into two breeding complexes and a growing complex in the region of Vysočina, Czech Republic and screened for the presence of Cryptosporidium using staining methods and genotyping (SSU rRNA). Cryptosporidium oocysts were detected by microscopy in the faeces of 21.1% of the samples (87/413). Sequence analyses and RFLP identified C. suis in 44, Cryptosporidium pig genotype II in 23 and C. muris in 2 samples. No mixed infections were found.Pigs under 7 weeks of age were infected with C. suis only. Cryptosporidium pig genotype II was found in animals from 7 weeks of age. No relationship was found between diarrhoea and any Cryptosporidium infection in any of the different age groups (P < 0.05). The pre-weaned pigs shed significantly more Cryptosporidium oocysts than older pigs and it was associated with C. suis infection.     

Phenotypic and genotypic characterization of Prototheca zopfii in a dog with enteric signs

This is a case report of enteric protothecosis caused by Prototheca zopfii in an eight-year-old male mixed breed dog with a history of chronic bloody diarrhea, loss of appetite and weight loss. Algae were isolated from rectal scrapings in defibrinated sheep blood agar and dextrose Sabouraud agar. Cytological evaluation showed the presence of globular and cylindrical organisms with a defined capsule and variable number of endospores, characteristic of the genus Prototheca, in the rectum of the animal. Scanning electron microscopy of P. zopfii strains at different development stages confirmed the diagnosis of algal infection. Molecular identification using a conserved 18S rDNA gene sequence determined that the strain belonged to genotype 2. This report describes success on treatment of canine protothecosis, diagnosed based on clinical, cytological, microbiological, scanning electron microscopy and genotypical findings.     

An increased incidence of mastitis caused by Prototheca species and Nocardia species on a farm in São Paulo,Brazil

Mastitis caused by Prototheca spp or Nocardia spp is considered to be difficult to treat. Both microorganisms are contaminants commonly found in soil. The occurrence of mastitis caused by these agents was studied in a particular dairy farm. In this herd, the animals were kept at pasture overnight and during daytime were brought to a pen where they were fed. This pen accumulated mud and faeces, particularly in the rainy season. During milking, pre-dipping of the teats was performed with an iodide solution, but they were not washed, so a layer of soil and faeces remained which may have contaminated the milking equipment. The herd comprised 91 lactating animals and 47 dry cows. For microbiological examination, 107 milk samples were collected from lactating cows and 186 samples of mammary secretions from the dry cows. Prototheca spp were isolated from 14.55% of the milk samples and Nocardia spp from 4.55%. Prototheca spp were isolated from 8.06% of the secretion samples from dry cows and Nocardia spp were isolated from 2.15% samples. The high occurrence of mastitis due to these environmental agents reflects the problem of keeping animals in muddy pastures and pens, and the defective pre-milking hygiene for the teats.     

Detection of a novel hemoplasma based on 16S rRNA gene DNA in captive and free-ranging capybaras (Hydrochaeris hydrochaeris)

Two different species of hemoplasmas, Mycoplasma coccoides and M. haemomuris, are known to infect small rodents such as mice and rats. However, there are no previous reports of hemoplasma infection in capybara (Hydrochaeris hydrochaeris). The aim of our study was to determine whether these hemoplasmas might infect capybaras from Southern Brazil. Blood samples from 31 animals: 10 captive and 21 free-ranging capybaras were collected and packed cell volume and total plasma protein were measured. DNA was extracted and PCR assays for M. coccoides and M. haemomuris were performed. Using the M. coccoides-PCR assay 64% of the capybaras were positive, 80% free-ranging and 30% from captive animals. The prevalence of infection between the groups was significantly different (p = 0.001). Sequencing of the nearly entire 16S rRNA gene from the positive samples suggested a novel hemoplasma isolate with identity of 92% with M. coccoides and 86% with M. haemomuris. All capybara samples were negative for M. haemomuris infection. DNA of a housekeeping gene was successfully amplified from all samples. This is the first evidence of a hemoplasma infection in capybaras.     

贵州某野生动物园长臂猿流感病毒、支原体及巴氏杆菌混合感染的诊治

为弄清贵州某野生动物园长臂猿死亡的原因,本试验采用流行病学调查、临床症状观察、病理剖检诊断和RT-PCR检测确诊等方法,对该野生动物园发病长臂猿进行了诊断。试验结果显示,流行病学调查、剖检病理初步诊断该野生动物园长臂猿疑似流感病毒、支原体和细菌混合感染,RT-PCR/PCR检测确诊发病长臂猿为流感病毒、支原体混合感染,细菌分离培养、生化特性鉴定和动物致病性试验确诊为多杀性巴氏杆菌感染。结果表明造成该野生动物园长臂猿发病死亡的原因为流感病毒、支原体和多杀性巴氏杆菌混合感染。根据诊断及药敏试验结果,制定出了免疫预防及治疗措施。     

Clinicopathological findings, molecular detection and characterization of Babesia gibsoni infection in a sick dog from Italy

A 4-year-old intact female American Pit Bull Terrier from Italy descendant of an American-born bitch was evaluated for anorexia, lethargy, weakness, and intermittent vomiting. On physical examination, the dog was dehydrated, had pale mucous membranes, hunched posture and abdominal pain. A moderate anemia was observed. Splenomegaly and hyperechoic regions suspected as infarcts in the spleen were seen on abdominal ultrasound. Based on the suspicion of splenic torsion, splenectomy was performed. After surgery, the clinical condition deteriorated. A follow-up complete blood count demonstrated severe macrocytic normochromic anemia with evidence of marked regeneration, left shift neutrophilia, monocytosis and marked thrombocytopenia. Blood smear evaluation revealed single to multiple, variable sized (1–3 μm in diameter), and round to oval to band-like piroplasms within many red blood cells consistent with small form Babesia spp. or Theileria spp. A partial segment of the 18S rRNA gene was amplified and the PCR product was analyzed by direct sequencing. The nucleotide sequence was completely identical to that of Babesia gibsoni present in GenBank^®. This is the first molecular detection and characterization of B. gibsoni infection in a sick dog from Italy.     

Role of pet dogs and cats in the transmission of helminthic zoonoses in Europe, with a focus on echinococcosis and toxocarosis

The close emotional tie between people and companion animals is a beneficial relation known as the human-animal bond. However, pet dogs and cats can play an important role in the transmission of helminthic zoonotic agents such as the tapeworms Echinococcus and the roundworms Toxocara which are directly transmitted from pets to the human environment without the involvement of vectors or intermediate hosts. In humans, echinococcosis has emerged in Europe and toxocarosis is still persisting in large endemic areas despite the availability of highly efficient anthelminthics for dogs and cats. Ecological changes significantly contributed to these trends: the high wild fox populations and the high density of freely roaming dogs and cats maintain a permanent infection pressure of these and other parasites. Further, the establishment of urban recreational environments closer to natural ecological systems boosted vole populations that represent urban reservoirs for zoonotic helminths. A good understanding of the parasites’ biology and epidemiology including the transmission to humans is required for planning and implementing effective prevention strategies. The continuous education of veterinarians and the information of the pet owners by providing uniform recommendations are of priority importance. A close collaboration between veterinary and public health professionals in a ‘One Health’ concept is required.     

Isolation and immunological detection of Mycoplasma ovipneumoniae in sheep with atypical pneumonia, and lack of a role for Mycoplasma arginini

Mycoplasma ovipneumoniae NCTC 10151(T) and four new isolates from UK sheep flocks were compared. Only glucose and pyruvate were used as energy sources by the five strains: glucose was the best energy source for the type strain, pyruvate supported better growth of the new strains. Whole cell protein patterns and antigenic profiles showed high similarity between all five strains. The new isolates fell into two groups in ELISA tests. Serum samples from 30 pneumonic sheep were assessed for M. ovipneumoniae infection and Mycoplasma arginini co-infection. Fourteen (out of 30) serum samples were positive for M. ovipneumoniae both by ELISA and immunoblotting. Twelve antigenic proteins of M. ovipneumoniae were detected in infected serum samples: the antigen patterns were unique, with between one and at least seven occurring in any one sample. All serum samples were designated as negative for M. arginini antibodies by both ELISA and immunoblotting.     

Study of the local immune response to Fasciola hepatica in the liver and hepatic lymph nodes of goats immunised with a peptide of the Sm14 antigen

The nature of the local immune response was assessed studying the distribution of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes, IgM⁺ B cells, IL-4⁺ and IFN-γ⁺ cells in the liver and hepatic lymph nodes (HLN) of goats immunised with a synthetic peptide of the Sm14 antigen from Schistosoma mansoni and challenged with Fasciola hepatica. A morphometric study of HLN was also carried out in order to evaluate the hyperplasia of lymphoid follicles. Despite the decrease in fluke burdens found in the immunised group (45.9%) respect to the infected control group, this difference was not statistically significant due to the high individual variability. In liver, a significant increase of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes was found in the infected groups respect to the uninfected control and in the infected control respect to the immunised group. HLN showed a significant enlargement due to the hyperplasia of lymphoid follicles and infiltration of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes in both infected groups respect to the uninfected control, with no significant differences between the infected control and immunised group. IFN-γ⁺ lymphoid cells was absent or very occasional in HLN where the number of IL-4⁺ cells was higher than that of IFN-γ, suggesting a polarized Th2 response in immunised and in infected control group.     

Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccine formulated with saponin or selected adjuvants

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.     

Effects of a standardized purified dry extract from Echinacea angustifolia on proliferation and interferon gamma secretion of peripheral blood mononuclear cells in dairy heifers

This study was performed to ascertain whether a standardized extract from Echinacea angustifolia (Polinacea™) affects proliferation and interferon gamma (IFN-γ) secretion in bovine peripheral blood mononuclear cells (PBMC).PBMC from six Holstein heifers were incubated with 0, 6.3, 20, 60, or 180 μg/ml of the tested compound. Proliferation was stimulated by concanavalin A (ConA) or pokeweed-mitogen (PWM). Secretion of IFN-γ was stimulated by ConA.All concentrations of Polinacea™ exerted a mitogenic effect. With respect to control PBMC (0 μg/ml), the lowest and highest increase of proliferation were observed with Polinacea™ at 6.3 (2-fold increase) or 180 (10-fold increase) μg/ml, respectively. Polinacea™ at 180 μg/ml reduced ConA-driven proliferation, whereas at 20 and 60 μg/ml improved proliferation of PWM-stimulated PBMC. IFN-γ secretion was not affected. In conclusion, Polinacea™ modulates bovine PBMC proliferation, and deserves to be tested in vivo to define conditions that may benefit from its utilization.