Genetic diversity of Streptococcus equi subsp. zooepidemicus isolated from horses

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic and zoonotic pathogen of horses. In this study, genetic intraspecies variability of SEZ obtained mainly from respiratory and genital samples of horses was investigated by analysis of the 16S–23S rRNA intergenic spacer region (ISR) and of the 16S rRNA gene. 16S–23S ISR rRNA type A1 was predominant, although a high rate of multiple products (30.5%) was obtained. Phylogenetic analysis of the 16S rRNA gene detected three genogroups (I, II and III). 16S rRNA variable regions V1 and V2 are the most important regions for evaluating SEZ intraspecies variability, but at least V1-V5 regions should be considered to avoid mistakes. Analysis of all 16S rRNA sequences available in databases assigned human SEZ to groups I and III but not to group II. These results show a high genetic variability in SEZ collected from different specimens of horses from various regions of Italy.     

Disseminated Rhodococcus equi infection in dromedary camels (Camelus dromedarius)

Rhodococcus (R). equi, a recognized pathogen in horses, is emerging as a human opportunistic pathogen, especially in immunocompromized people. It affects also New World camelids, but there are no reports of R. equi infection in Old World camelids yet. Four cases of disseminated R. equi infection in adult breeding dromedaries occurred at one camel farm near Dubai within 16 months of each other. At necropsy the lungs were diffusely consolidated with large caseous areas. Histology revealed severe suppurative to necrotising pneumonia with multiple encapsulated abscesses. Immunohistochemistry enabled the detection of 15- to 17-kDa antigens (VapA) of R. equi in the lung sections. High numbers of R. equi were isolated from the lung lesions as well as from liver, spleen and mediastinal lymph nodes, indicative of septicaemia. The isolated strains were PCR-positive for the specific virulence plasmid (VapA-Gen) of R. equi, indicating virulent strains and containing an 85-kb type I plasmid. This is the first report of disseminated R. equi infection in Old World camelids. Since adult camels in general do not suffer from bacterial caused pneumonia (except tuberculosis), this is a new emerging disease for camels.     

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.     

Multiplex polymerase chain reaction for identification and differentiation of Streptococcus equi subsp. zooepidemicus and Streptococcus equi subsp. equi

The closely related streptococcal species Streptococcus equi subsp. zooepidemicus and S. equi subsp. equi were identified by polymerase chain reaction using oligonucleotide primers designed according to species-specific parts of the superoxide dismutase A encoding gene sodA. A further differentiation of both subspecies could be performed by amplification of the genes seeH and seeI encoding the exotoxins SeeH and SeeI, respectively, which could be detected for S. equi subsp. equi but not for S. equi subsp. zooepidemicus. A further simplification of the identification and differentiation of both subspecies was conducted by sodA-seeI multiplex polymerase chain reaction.     

Isolation and further characterization of phase variants of Streptococcus equi subsp. zooepidemicus

In the present study the soft agar technique was used to isolate phase variants of S. equi subsp. zooepidemicus-cultures isolated from infections of horses. The phase variants were characterized by a compact or diffuse colony morphology in this media. The variants could be cultivated separately and further characterized genotypically by RAPD analysis and by macrorestriction analysis of their chromosomal DNA by pulsed-field gel electrophoresis, indicating the identity of both strains of each pair. The diffuse colony variants grew uniformly turbid after cultivation in fluid media, did not haemagglutinate rabbit erythrocytes, and displayed a reduced surface hydrophobicity in hexadecane and phenyl-sepharose adherence tests. The compact colony variants generally grew as sediment with clear supernatant in fluid media, haemagglutinated rabbit erythrocytes and showed an enhanced surface hydrophobicity in both hydrophobicity tests. The presented soft agar technique allowed a demonstration of phase variation of S. equi subsp. zooepidemicus and a subsequent isolation of the variants. This might be an important prerequisite to understanding the pathogenic importance of phase variation among isolates of this bacterial species.     

Isolation and characterization of Mycoplasma arginini from camels (Camelus dromedarius) with pneumonia

Post-mortem examinations of 100 camels with pneumonic lesions were made at a local abattoir for Mycoplasma species. Sixteen isolates with indistinguishable biochemical and immunological characters were identified. The biochemical profile of these isolates showed that they were sensitive to digitonin, negative for urease production, glucose fermentation, and phosphatase activity but were positive for arginine hydrolysis. The identity of these isolates was further confirmed by disk growth inhibition test using a panel of specific antisera against selected reference Mycoplasma spp. Based on the biochemical profile and growth inhibition results, the camel isolates were identified as M. arginini. The pathological findings associated with M. arginini isolation consisted mostly of chronic interstitial pneumonia. The isolation rate of M. arginini from these specimens was 8.8%. These results suggest that the role of M. arginini in pneumonia in camels should be explored in greater detail.     

Productivity and health of camels (Camelus dromedarius) in Somalia: Associations with trypanosomosis and brucellosis

Summary In Somalia, one of the world’s largest dromedary populations of about 5–3 million animals are kept by nomadic pastoralists under traditional management. Interest in the development potential of camel herds in the semi-aird areas of central Somalia initiated an investigation to determine the productivity of herds, their major diseases and likely associations among these parameters. Using a systems approach, data were collected for herd production parameters, environmental factors, management and production systems, and health variables. One thousand and thirty nine camels in 33 herds were studied in the central regions of Somalia.Trypanosoma evansi prevalence ranged from 1·7% in bloodsmears to 56·4% using enzyme-linked immunosorbent micro-assay (micro ELISA). Seroprevalence for brucellosis was determined as 1·9% by the standard agglutination test (SAT) and 0·3% by the complement fixation test (CFT). Using multiple regression, 15% of the total variation of the general fertility rate was explained by the results of the microhaematocrit centrifugation technique (MHCT) and the microELISA forT. evansi, CFT results for brucellosis, herdsize, and young stock death rate. Among herd production variables, herd size differd significantly for different management units. Young stock death rates, as well as general fertility rates varied in the ecological subzones with a marked effect in the zones labeled “Inland”. Various other associations were noted among demographic, husbandry and disease variables. The importance of trypanosomosis and brucellosis to the productivity of herds and measures to control their limiting effects on production were discussed.

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Résumé En Somalia existe una de las poblaciones más numerosas de camellos en el mundo. Cerca de 5·3 millones de animales pertenecen a pastores nómadas, los que los manejan bajo sistemas tradicionales. Debido al interés de conocer el potencial productivo de los hatos en las regiones semiáridas de Somalia Central, se inició una investigatión acerca de las principales enfermedades y la asociación entre éstas y la productividad en general. Utilizando un enfoque de sistemas, se colectaron resultados de parámetros productivos, factores ambientales, sistemas de manejo y producción y variables sanitarias. Se estudiaron 1,039 camellos en 33 hatos. La prevalencia deTrypanosoma evansi fluctuó entre 17% en frotis de sangre, hasta 56.4% utilizando microELISA. La seroprevalencia de brucelosis fue de 1·9% con la prueba de aglutinación y de 0·3% con la prueba de fijación del complemento. Usando regresiones múltiples, 15% de la variación total de la tasa de fertilidad general, se pudo explicar relacionando ésta con los resultados de la técnica del microhematocrito utilizando centrifuga, de microELISA paraT. evansi, de fijación del complemento para brucelosis, con el tama?o del hato y con la tasa de mortalidad en animales jóvenes. Entre las variables de producción del hato, el tama?o del mismo difirió significativamente para las diferentes unidades de manejo. Las tasas de mortalidad de animales jóvenes y las de fertilidad, variaron de acuerdo a las subzonas ecológicas, con un efecto marcado en las zonas mediterraneas. Se presentaron también asociaciones entre variables demográficas, de manejo y sanitarias. Se discute la importancia de la tripanosomiasis y de la brucelosis, en relación con la productividad del hato y las medidas para controlarlas, minimizando así su efecto sobre la producción.

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Résumé L’une des plus grandes populations de dromadaires au monde vit en Somalie où l’on compte environ 5,3 millions de têtes élevées par des pasteurs nomades selon le mode traditionnel. L’intérêt pour le développement potentiel du cheptel camelin dans la zone centrale semi-aride du pays, a été à l’origine d’une enquête pour déterminer la productivité des troupeaux, les affections essentielles et vraisemblablement les points de convergence entre ces différents paramètres. Par le biais d’une approche systématique, des données ont été rassemblées sur les facteurs de production, le milieu, la gestion et les modes de faire-valoir et les variables d’ordre sanitaire. 1 039 dromadaires, répartis en 33 troupeaux, ont fait l’objet de cette étude dans la région centrale de la Somalie. La prévalence deTrypanosoma evansi a varié de 1,7 p. 100 avec les étalements de sang à 56,4 p. 100 par l’emploi du test micro-ELISA (micro-test d’immunoabsorption enzymatique). La séroprévalence de la brucellose a été évaluée à 1,9 p. 100 par le test standard de la séroagglutination et à 0,3 p. 100 par la fixation du complément. Enfin par l’emploid d’une équation de régression multiple, 1,5 p. 100 de la variation totale de la fertilité au sens large ont pu être expliqués grace aux données recueillies avec la technique de centrigufation au microhématocrite et le micro test ELISA pour la recherche deTrypanosoma evansi, par la fixation du complément pour la brucellose, la taille des troupeaux et le taux de mortalité chez les jeunes. Au nombre des variables de la production des troupeaux, la taille différait de fa?on significative en fonction des differentes unités de gestion. Les taux de mortalité ches les jeunes, de même que ceux de la fertilité en général ont varié dans les sous-régions écologiques mais avec un effect marqué dans les zones qualifiées d’intérieures (Inland). Différentes associations sont également perceptibles au niveau de certaines variables telles que la demographie, la gestion et la pathologie. L’importance de la trypanosomose et de la brucellose au regard de la productivité des troupeaux ainsi que les mesures de contr?le pour en limiter les conséquences sur la production font l’objet d’une discussion.

     

Isospora orlovi infection in suckling dromedary camel calves (Camelus dromedarius) in Kenya

Outbreaks of isosporosis in young suckling dromedary camel calves (Camelus dromedarius) in Dubai, UAE and in Kenya were recently described. In the former outbreak the pathogen was shown to be Isospora orlovi by morphological features and was later characterized molecularly. In the present study, we have made a longitudinal investigation of 159 suckling dromedary calves < or =12 weeks of age belonging to 8 ranched camel herds (M1) in Northern Kenya. The study was carried out during 18 months. In three of the herds frequent samples were taken irregularly every 1-6 weeks. All calves < or =12 weeks of age present in the respective herds were sampled during the visits. In addition, 91 calves of the same age group but belonging to 42 pastoral herds (M2) in Northern Kenya were point sampled at convenience. Faecal samples from each calf were taken and the faeces were investigated for coccidia. Samples found with coccidian oocysts were suspended in a 2% potassium dichromate solution. Isospora sp. was identified and samples with relatively high numbers of Isospora sp. were analysed molecularly. The SSU rRNA gene and internal transcribed spacer 1 (ITS1) were amplified with primers complementary to conserved regions of the SSU rRNA gene in eukaryotes as well as a conserved part of the 5.8S rRNA gene of Eimeria. A relatively high number of the calves exhibited diarrhoea, 30.2% and 41.8% in the M1 and M2 herds, respectively. Isospora sp. was only found in diarrhoeic calves or in calves convalescent from recent scouring periods. No calf >8 weeks of age was found to be excreting Isospora sp. The parasite was only found in calves < or =4 weeks of age in the M1 herds and in the M2 herds in calves <8 weeks of age. Of the M1 and M2 calves exhibiting diarrhoea, 20.8% and 26.3% excreted Isospora sp., respectively. Morphologically the Isospora sp. was similar to I. orlovi and sequence analysis of the SSU rRNA gene from four Kenyan isolates (unfortunately only from the pastoral herds, M2) and ITS 1 segments from three of the isolates from Kenya and one from Dubai, confirmed that the Isospora isolates belonged to the species I. orlovi, and that the sequences were similar to the Dubai isolates.     

马源马链球菌兽疫亚种新疆株的分离鉴定及遗传特性分析

为了解马源马链球菌兽疫亚种（S.zooepidemicus）新疆分离株马链球菌兽疫亚种类M蛋白（SzM）基因的分子进化与变异情况,为该菌引起的感染性疾病的防控提供依据,本试验对新疆地区某马场采集的病马淋巴结样品进行病原菌的分离培养和生化鉴定,并对分离菌株进行药物敏感性试验。根据已发表的马源SzM基因序列设计引物,对其SzM基因进行PCR扩增及序列测定。将获得的SzM序列与GenBank中不同动物源马链球菌兽疫亚种分离株序列进行同源性比对和遗传进化分析。结果显示,分离得到了一株革兰氏阳性链球菌,将其命名为马链球菌兽疫亚种ZMSY15-1。药物敏感性试验结果表明,分离菌株对青霉素、磺胺嘧啶钠耐药,对其他14种药物均敏感。序列分析结果显示,马链球菌兽疫亚种ZMSY15-1与国内外不同动物源分离株SzM基因氨基酸同源性为56.0%~70.0%。遗传进化分析结果显示,这些菌株可分为4个群。马链球菌兽疫亚种ZMSY15-1与猪源分离株SzM蛋白的氨基酸同源性为59.9%,分别属于2个不同的群,其与美国马源分离株NH55426亲缘关系最近。本试验结果可丰富国内马源马链球菌兽疫亚种SzM基因的信息数据,为马链球菌兽疫亚种的致病机制研究和预防控制提供参考依据。     

Mycoplasma detection and isolation from one-humped camels (Camelus dromedarius)

In scientific literature, a small amount of information is found concerning mycoplasmosis in camel species. Mycoplasma (M.) arginini, Acholeplasma (A.) laidlawii, and Acholeplasma oculi have been reported to be isolated from these host species. Serologically positive results have been reported for Mycoplasma mycoides subsp. mycoides SC type, Mycoplasma capricolum subsp. capripneumoniae, and M. mycoides subsp. capri. The aims of this study were to detect, isolate, and identify mycoplasmas from camels (Camelus dromedarius). Initially, saliva and ear smears plus conjunctival and vaginal secretions were taken from five female animals, but only conjunctival secretions in three male animals, all belonging to the same farm. An unknown mycoplasma was isolated from one of the vagina samples. Additionally, another unknown and uncultured mycoplasma was detected with molecular biology in the same sample. In the second stage, 23 vaginal secretions were taken from the same farm plus another secretion from a different one. Ten isolates of the same unknown and previously isolated mycoplasma were detected, nine of them recovered from the vagina of female camels. Some mycoplasmas have been related to reproductive disorders; however, there is no evidence that the isolated mycoplasmas are related to such disorders.     

Application of the California mastitis test in intramammary Streptococcus agalactiae and Staphylococcus aureus infections of camels (Camelus dromedarius) in Kenya

A study was conducted on 207 lactating camels in six herds in Kenya to evaluate the California mastitis test (CMT) for the detection of intramammary infections (IMIs) caused by Streptococcus agalactiae and Staphylococcus aureus and to investigate the prevalence of both the pathogens in the camel udder. IMI with S. agalactiae was found in 12% of all camels sampled. IMI with S. aureus was present in 11% of all camels sampled. The herd-level prevalence of IMI varied between 0 and 50% for S. agalactiae and between 0 and 13% for S. aureus. Longitudinal observations over 10–12 months confirmed persistent infections for both pathogens. Observations in one herd suggested that camel pox was a contributing factor in spreading and exacerbating S. agalactiae udder infections.

The CMT had quarter-level sensitivities of 77 and 68% for S. agalactiae and S. aureus in camels, respectively. The CMT specificities were 91% for both the pathogens.     

Physiological change in camel milk composition (Camelus dromedarius) 1. Effect of lactation stage

The change in the composition of camel milk in four dromedaries was studied by including the common measured parameters: protein, total fat, lactose, main minerals (calcium, phosphorus, and iron), and vitamin C. The fat matter varied from 4.34% to 7.81% with a slight decrease all along the lactation and a minimal value at the 14th week corresponding to the lactation peak. Those variations were less important for protein content (from 2.58% to 3.64%), but the minimal value was observed at the 14th week also. The lactose varied slightly around its mean of 3.46%. The vitamin C concentration varied from 48 to 256 mg/l with a tendency of increasing all along the lactation. Calcium and phosphorus concentrations were quite parallel and their ratio Ca/P was constant. The minimal values (1.43 g/l for calcium and 1.16 g/l for phosphorus) were observed at the beginning of the lactation. The iron concentrations varied around the mean of 1.73 mg/l.     

Actinomycosis in a one-humped camel (Camelus dromedarius)

An actinomycotic granuloma caused by Actinomyces viscosus is reported in a dromedary camel. Two hard, cutaneous, large granulamatous nodules were present on both sides of the postero-ventral side of the mandible exhibiting exudation and necrosis. After radical excision of the lesion, the daily treatment with penicillin-streptomycin combination was continued for 4 weeks. About 8 and 24 weeks from the initial treatment, no new nodules were noticed.