东北虎源等孢球虫虫种鉴定及其生物学特性与药物防治研究
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摘要
珍稀观赏动物的球虫疾病报道近年来逐渐增多。笔者对黑龙江省某动物园的92只猫科动物进行了球虫感染调查并进行了六种粪检法检测效力的测定,结果teleman法+饱和盐-糖溶液漂浮法为最佳的检测方法,其球虫卵囊检出率为17.39%(16/92),其中幼龄动物感染明显多于成年动物。
对球虫卵囊培养后进行形态学观察:未孢子化卵囊多数呈卵圆形,平均长为45.875μm,平均宽为34.775μm,形状指数为1.19。卵壁光滑没有颜色。卵膜共分两层,外层薄而无色,内层厚呈黑色。无卵膜孔,无极粒,无残体。卵囊孢子化后,胚囊分裂成为两个孢子囊,每个孢子囊中含有四个子孢子,残体清晰可见。孢子囊呈椭圆形,平均长为26.875μm,平均宽为22.375μm,形状指数为1.25。提取虫体基因组DNA,并根据GenBank上发表的猫等孢球虫核糖体内转录间隔区Ⅰ(Cystoisospora felis internal transcribed spacer 1, ITS1)序列设计引物,对目的片段进行PCR扩增和序列测定。应用DNAStar软件,将长为389bp的目的片段与Genbank中发表的19种球虫相应的ITS1序列进行同源性比较,序列分析显示东北虎源球虫与其它19种球虫相应序列同源性为38.11%～98.71%之间,其中与等孢球虫的同源性为55.64%～98.71%之间。综合对东北虎源球虫形态学观察、PCR扩增其ITS1序列并进行序列分析,构建分子进化树及系统发生关系的分析以及动物感染实验,确定东北虎源球虫为等孢属球虫,并且与猫等孢球虫亲缘最近。
应用单卵囊分离技术,获得东北虎源等孢球虫纯种卵囊。分别给猫感染30、103、104个孢子化卵囊,观察猫的排卵囊规律,包括潜隐期、显露期、排卵囊高峰期及每克粪便中卵囊数(OPG值)。通过对比,验证了潜隐期、显露期、OPG值与感染剂量存在相关关系,即感染剂量越小,潜隐期越长,显露期越长,OPG值越小;反之亦然。感染30个卵囊的猫无明显临床病征;感染103个卵囊的猫腹泻出现少量血便;感染104个卵囊的猫水样腹泻后出现大量血便。
感染103与104个孢子化卵囊的猫完成卵囊排放,于10d后再次感染103个孢子化卵囊,观察猫的排卵囊规律,包括潜隐期、显露期、排卵囊高峰期及OPG值。结果表明感染强度与免疫力存在正相关关系,即首次感染强度较大组产生的免疫记忆较强,再次感染相同剂量卵囊后,其卵囊排出数量少,表现出的病症也相对较轻。
妥曲株利和莫能菌素分别按0.4ml·kg-1bw和15mg·kg-1bw喂猫并于当日接种104个孢子化卵囊,首次用药后24h再给药一次。于感染后三天(PID3)开始,用饱和糖-盐水溶液漂浮法检查粪便,观察猫的排卵囊规律,包括潜隐期、显露期、排卵囊高峰期及OPG值。结果表明两预防组均无卵囊检出,妥曲株利与莫能菌素给药后均可有效地预防等孢球虫感染。
给猫感染104个孢子化卵囊,于卵囊始现日将妥曲株利和莫能菌素分别按0.4ml·kg-1bw和15mg·kg-1bw喂猫,首次用药后24h再给药一次。结果给药后,两治疗组的卵囊排出强度快速下降。与莫能菌素相比,百球清表现出更强、更快的杀虫和抑虫作用,其治疗效果更为优秀。
利用妥曲株利和莫能菌素进行药物预防和治疗实验的猫,于粪中卵囊最早消失的时间延后10d,再次感染104个孢子化卵囊,于PID3开始,用饱和糖-盐水溶液漂浮法检查粪便,观察猫的排卵囊规律,包括潜隐期、显露期、排卵囊高峰期及OPG值。结果表明:两药物预防组的猫再次感染卵囊后,其卵囊排出强度与正常一次感染组相似,远大于正常二次感染组。这说明猫在药物预防后感染卵囊未获得免疫记忆,因此再次感染球虫后卵囊的排出强度没有降低;而两治疗组的猫再次感染卵囊后,其卵囊排出强度远小于正常二次感染组。与莫能菌素相比,妥曲株利治疗后的猫产生了较强的免疫力,再次感染后卵囊的排出强度较小。
There are more and more reports about infections of Isospora oocysts in valuable ornamental feline animals in recent years. A total of 92 fecal samples of mufti-feline animals were collected from a zoo in HeiLongJiang province and were inspected for coccidiosis. After screening six laboratory examinatins of dung. detection rate of coccidian was 17.39%(16/19) by teleman method and saturated salt - sugar solution floating method which was the optimum and the youngers is more than the olders in infection of coccidiosis.
Observation of coccidial morphology was taken in cultivation of oocysts. Unsporuled oocysts most are orbicular-ovate, its mean length is 45.875μm and mean width is 34.775μm, the shape factor is 1.19. The paries of oocyst is smooth and no color and oocyst membrane has two layer which stratum internum is thick and black but ectosome is thin and achromatous. The oocyst has no fenestrated membrane and polar granule also residual body. When oocyst had been sporuled, the embryo sac began to split into two sporocysts which has four sporozoites each and residual body is clear. The sporosac is oval-shap, its mean length is 26.875μm and mean width is 22.375μm, the shape factor is 1.25. After Gotten the genomic DNA, Specie-specific PCR primer was designed which got at the Cystoisospora felis internal transcribed spacer 1. Done the PCR amplification and sequencing. By the DNAStar, compared the fraction of 389bp with the other 19 fractions got from Genbank, the consequence is that the cognation among them is 35.97%~98.71%, to Isospora is 55.64%~98.71%. From the obversion of the configuration, the result of PCR and the comparison of the ITS1 fractions and the result of animal infection, we can confirm that the coccidia oocysts from the Siberian tiger is the Isospora coccidia oocysts and It is closer to Isospora felis than others.
Pure Isospora oocysts from tiger were obtained through separation and cats were orally inoculated with 30 and 103 and 104 sporuled oocysts each group. The regularity of oocysts excretion contained the prepatent period, the patent period, the peak period, the number of oocyst in per gram feces(OPG value) and clinic symptom. The results indicates that the prepatent period, the patent period and the OPG value is correlated with the number of oocyst,namely, the smaller the number of oocyst, the longer theprepatent period, the longer the patent period and the smaller the infection intensity; and vice versa. During the course of infection, the cats inoculated with 30 oocysts did not present any conspicuous symptoms, whilst the cats inoculated with 103 oocysts showed slight symptoms or developed diarrhea and bloody stools and the cats inoculated with 104 oocysts were more severe.
The two groups which had been inoculated with 103 and 104 sporuled oocysts finished their oocysts excretion. From then on, give 104 sporuled oocysts 10 days later. Got the regularity of oocysts excretion contained the prepatent period, the patent period, the peak period, the number of oocyst in per gram feces. The results indicates that the immunity is correlated with the infection intensity, namely, the bigger the infection intensity, the better the immunologic memory, the smaller the number of oocysts excretion next infection and the slighter the clinic symptom; and vice versa.
Cats were treated with Toltrazuril solution(Baycox) by 0.4ml·kg-1bw and monensin premix by 15mg·kg-1bw, respectively. And given 104 sporuled oocysts at the same day. Next medication was taken in 24 hours later and Got the regularity of oocysts excretion contained the prepatent period,the patent period, the peak period, the number of oocyst in per gram feces from PID3. The results demonstrated that the both groups were negative and the two medicine were efficient in preventing cats from isosporosis.
Cats which had been had been inoculated with 104 sporuled oocysts were treated with Toltrazuril solution(Baycox) by 0.4ml·kg-1bw and monensin premix by 15mg·kg-1bw, respectively, at the frist day of oocysts excretion. Next medication was taken in 24 hours later. The results demonstrated that the intensity of oocysts excretion were descendent markedly in both groups. Comparing with monensin premix, Toltrazuril solution(Baycox) had bigger and faster effects in anthelminthic and inhibition, so it is more efficient in treating isosporosis of cats.
Cats which had been given Toltrazuril solution(Baycox) and monensin premix for prevention and therapia were inoculated with 104 sporuled oocysts again and Got the regularity of oocysts excretion contained the prepatent period, the patent period, the peak period, the number of oocyst in per gram fecesfrom PID3. The results demonstrated that the intensity of oocysts excretion in prev group was almost same as the cats inoculated with 104 sporuled oocysts only once and were bigger than the cats inoculated with 104 sporuled oocysts twice. Because of the lack of immunologic memory in the process of drug prophylaxis, the intensity of oocysts excretion after second infection were invariant.; the intensity of oocysts excretion in cur group was far smaller than the cats inoculated with 104 sporuled oocysts twice. Compared with monensin premix, the group cured by Baycox had a bigger immunity and got smaller intensity of oocysts excretion in next infection.
对球虫卵囊培养后进行形态学观察:未孢子化卵囊多数呈卵圆形,平均长为45.875μm,平均宽为34.775μm,形状指数为1.19。卵壁光滑没有颜色。卵膜共分两层,外层薄而无色,内层厚呈黑色。无卵膜孔,无极粒,无残体。卵囊孢子化后,胚囊分裂成为两个孢子囊,每个孢子囊中含有四个子孢子,残体清晰可见。孢子囊呈椭圆形,平均长为26.875μm,平均宽为22.375μm,形状指数为1.25。提取虫体基因组DNA,并根据GenBank上发表的猫等孢球虫核糖体内转录间隔区Ⅰ(Cystoisospora felis internal transcribed spacer 1, ITS1)序列设计引物,对目的片段进行PCR扩增和序列测定。应用DNAStar软件,将长为389bp的目的片段与Genbank中发表的19种球虫相应的ITS1序列进行同源性比较,序列分析显示东北虎源球虫与其它19种球虫相应序列同源性为38.11%～98.71%之间,其中与等孢球虫的同源性为55.64%～98.71%之间。综合对东北虎源球虫形态学观察、PCR扩增其ITS1序列并进行序列分析,构建分子进化树及系统发生关系的分析以及动物感染实验,确定东北虎源球虫为等孢属球虫,并且与猫等孢球虫亲缘最近。
应用单卵囊分离技术,获得东北虎源等孢球虫纯种卵囊。分别给猫感染30、103、104个孢子化卵囊,观察猫的排卵囊规律,包括潜隐期、显露期、排卵囊高峰期及每克粪便中卵囊数(OPG值)。通过对比,验证了潜隐期、显露期、OPG值与感染剂量存在相关关系,即感染剂量越小,潜隐期越长,显露期越长,OPG值越小;反之亦然。感染30个卵囊的猫无明显临床病征;感染103个卵囊的猫腹泻出现少量血便;感染104个卵囊的猫水样腹泻后出现大量血便。
感染103与104个孢子化卵囊的猫完成卵囊排放,于10d后再次感染103个孢子化卵囊,观察猫的排卵囊规律,包括潜隐期、显露期、排卵囊高峰期及OPG值。结果表明感染强度与免疫力存在正相关关系,即首次感染强度较大组产生的免疫记忆较强,再次感染相同剂量卵囊后,其卵囊排出数量少,表现出的病症也相对较轻。
妥曲株利和莫能菌素分别按0.4ml·kg-1bw和15mg·kg-1bw喂猫并于当日接种104个孢子化卵囊,首次用药后24h再给药一次。于感染后三天(PID3)开始,用饱和糖-盐水溶液漂浮法检查粪便,观察猫的排卵囊规律,包括潜隐期、显露期、排卵囊高峰期及OPG值。结果表明两预防组均无卵囊检出,妥曲株利与莫能菌素给药后均可有效地预防等孢球虫感染。
给猫感染104个孢子化卵囊,于卵囊始现日将妥曲株利和莫能菌素分别按0.4ml·kg-1bw和15mg·kg-1bw喂猫,首次用药后24h再给药一次。结果给药后,两治疗组的卵囊排出强度快速下降。与莫能菌素相比,百球清表现出更强、更快的杀虫和抑虫作用,其治疗效果更为优秀。
利用妥曲株利和莫能菌素进行药物预防和治疗实验的猫,于粪中卵囊最早消失的时间延后10d,再次感染104个孢子化卵囊,于PID3开始,用饱和糖-盐水溶液漂浮法检查粪便,观察猫的排卵囊规律,包括潜隐期、显露期、排卵囊高峰期及OPG值。结果表明:两药物预防组的猫再次感染卵囊后,其卵囊排出强度与正常一次感染组相似,远大于正常二次感染组。这说明猫在药物预防后感染卵囊未获得免疫记忆,因此再次感染球虫后卵囊的排出强度没有降低;而两治疗组的猫再次感染卵囊后,其卵囊排出强度远小于正常二次感染组。与莫能菌素相比,妥曲株利治疗后的猫产生了较强的免疫力,再次感染后卵囊的排出强度较小。
There are more and more reports about infections of Isospora oocysts in valuable ornamental feline animals in recent years. A total of 92 fecal samples of mufti-feline animals were collected from a zoo in HeiLongJiang province and were inspected for coccidiosis. After screening six laboratory examinatins of dung. detection rate of coccidian was 17.39%(16/19) by teleman method and saturated salt - sugar solution floating method which was the optimum and the youngers is more than the olders in infection of coccidiosis.
Observation of coccidial morphology was taken in cultivation of oocysts. Unsporuled oocysts most are orbicular-ovate, its mean length is 45.875μm and mean width is 34.775μm, the shape factor is 1.19. The paries of oocyst is smooth and no color and oocyst membrane has two layer which stratum internum is thick and black but ectosome is thin and achromatous. The oocyst has no fenestrated membrane and polar granule also residual body. When oocyst had been sporuled, the embryo sac began to split into two sporocysts which has four sporozoites each and residual body is clear. The sporosac is oval-shap, its mean length is 26.875μm and mean width is 22.375μm, the shape factor is 1.25. After Gotten the genomic DNA, Specie-specific PCR primer was designed which got at the Cystoisospora felis internal transcribed spacer 1. Done the PCR amplification and sequencing. By the DNAStar, compared the fraction of 389bp with the other 19 fractions got from Genbank, the consequence is that the cognation among them is 35.97%~98.71%, to Isospora is 55.64%~98.71%. From the obversion of the configuration, the result of PCR and the comparison of the ITS1 fractions and the result of animal infection, we can confirm that the coccidia oocysts from the Siberian tiger is the Isospora coccidia oocysts and It is closer to Isospora felis than others.
Pure Isospora oocysts from tiger were obtained through separation and cats were orally inoculated with 30 and 103 and 104 sporuled oocysts each group. The regularity of oocysts excretion contained the prepatent period, the patent period, the peak period, the number of oocyst in per gram feces(OPG value) and clinic symptom. The results indicates that the prepatent period, the patent period and the OPG value is correlated with the number of oocyst,namely, the smaller the number of oocyst, the longer theprepatent period, the longer the patent period and the smaller the infection intensity; and vice versa. During the course of infection, the cats inoculated with 30 oocysts did not present any conspicuous symptoms, whilst the cats inoculated with 103 oocysts showed slight symptoms or developed diarrhea and bloody stools and the cats inoculated with 104 oocysts were more severe.
The two groups which had been inoculated with 103 and 104 sporuled oocysts finished their oocysts excretion. From then on, give 104 sporuled oocysts 10 days later. Got the regularity of oocysts excretion contained the prepatent period, the patent period, the peak period, the number of oocyst in per gram feces. The results indicates that the immunity is correlated with the infection intensity, namely, the bigger the infection intensity, the better the immunologic memory, the smaller the number of oocysts excretion next infection and the slighter the clinic symptom; and vice versa.
Cats were treated with Toltrazuril solution(Baycox) by 0.4ml·kg-1bw and monensin premix by 15mg·kg-1bw, respectively. And given 104 sporuled oocysts at the same day. Next medication was taken in 24 hours later and Got the regularity of oocysts excretion contained the prepatent period,the patent period, the peak period, the number of oocyst in per gram feces from PID3. The results demonstrated that the both groups were negative and the two medicine were efficient in preventing cats from isosporosis.
Cats which had been had been inoculated with 104 sporuled oocysts were treated with Toltrazuril solution(Baycox) by 0.4ml·kg-1bw and monensin premix by 15mg·kg-1bw, respectively, at the frist day of oocysts excretion. Next medication was taken in 24 hours later. The results demonstrated that the intensity of oocysts excretion were descendent markedly in both groups. Comparing with monensin premix, Toltrazuril solution(Baycox) had bigger and faster effects in anthelminthic and inhibition, so it is more efficient in treating isosporosis of cats.
Cats which had been given Toltrazuril solution(Baycox) and monensin premix for prevention and therapia were inoculated with 104 sporuled oocysts again and Got the regularity of oocysts excretion contained the prepatent period, the patent period, the peak period, the number of oocyst in per gram fecesfrom PID3. The results demonstrated that the intensity of oocysts excretion in prev group was almost same as the cats inoculated with 104 sporuled oocysts only once and were bigger than the cats inoculated with 104 sporuled oocysts twice. Because of the lack of immunologic memory in the process of drug prophylaxis, the intensity of oocysts excretion after second infection were invariant.; the intensity of oocysts excretion in cur group was far smaller than the cats inoculated with 104 sporuled oocysts twice. Compared with monensin premix, the group cured by Baycox had a bigger immunity and got smaller intensity of oocysts excretion in next infection.
引文
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