马属动物毕氏肠微孢子虫研究进展

毕氏肠微孢子虫是一种引起人和动物腹泻的常见病原体,可通过食物和水源传播,具有重要的公共卫生意义。根据系统进化分析,已鉴定出近300个毕氏肠微孢子虫基因型,分为9个组群,组群1中的基因型大多数具有潜在人兽共患性;组群2~8中的基因型多数具有宿主特异性,部分也可感染人。研究表明,马属动物可感染37个毕氏肠微孢子虫基因型,具有一定人兽共患风险。论文就近年来感染马属动物的毕氏肠微孢子虫基因型、人兽共患风险、流行情况和致病性等进行综述。     

藏香猪毕氏肠微孢子虫感染情况和基因分型

采集了青海省和陕西省部分地区共计450份藏香猪新鲜粪便样品,基于小亚基核糖体RNA基因(small-subunit ribosomal RNA,SSU rRNA)的巢式PCR检测样品中毕氏肠微孢子虫(Enterocytozoon bieneusi)的感染情况,并分析其遗传多样性。结果显示,218份样品感染了毕氏肠微孢子虫,总感染率为48.4%(218/450);青海省所调查藏香猪的毕氏肠微孢子虫感染率为65.8%(154/234),显著高于陕西省的感染率29.6%(64/216)(P0.001);4个年龄段间藏香猪毕氏肠微孢子虫感染率差异显著(P0.001),其中哺乳仔猪感染率最高(92.7%),而成年猪感染率最低(9.2%)。序列分析发现,藏香猪存在5个SSU rRNA基因型(Type 1～Type 5),其中Type 1为优势基因型。对218份毕氏肠微孢子虫阳性分离株的多位点序列分型发现,分别有59,26,9和99份样品在MS1、MS3、MS4和MS7基因位点成功扩增,呈现18,4,4和4种单倍型,基于MS1、MS3和MS4等3个基因位点共形成6种多位点基因型(multi-locus genotypes,MLGs)。     

First report of Enterocytozoon bieneusi from dairy cattle in Argentina

Fecal specimens were obtained from a total of 70 dairy calves less than two months old on 11 municipalities in Buenos Aires, Argentina. After removal of fecal debris by sieving and sucrose flotation, specimens were subjected to PCR to detect the presence of Enterocytozoon bieneusi. PCR revealed a 14.3% of prevalence for E. bieneusi with 10 positive calves from 7 municipalities. Gene sequence analysis conducted in all samples positives by PCR revealed the presence of six genotypes; four previously reported in cattle as well as humans (D, I, J, and BEB4), one never reported in cattle before but previously reported in humans (EbpC), and one novel genotype (BEB10). These results constitute the first molecular characterization of E. bieneusi in Argentina, and suggest a potential risk of zoonotic transmission in this area.     

秦岭地区几种珍稀动物毕氏肠微孢子虫基因分型

为了评估秦岭地区珍稀野生动物的毕氏肠微孢子虫(Enterocytozoon bieneusi)的人兽共患风险,本研究采用PCR技术和多位点序列分型技术对秦岭地区5种珍稀动物毕氏肠微孢子虫的感染情况及基因型/亚型进行了分析。在采集的22份新鲜粪便中检测到7个阳性分离株,其中斑羚源5个,长颈鹿源1个,麂子源1个。基于ITS位点基因分型发现所有分离株均为人兽共患基因型,包括6个已报道的基因型D和1个尚未报道的D-new。多位点序列分型技术发现秦岭地区珍稀野生动物的毕氏肠微孢子虫存在遗传多样性,7个分离株在MS1、MS3、MS4和MS7位点分别有3、1、2、2个亚型。     

长春地区梅花鹿毕氏肠微孢子虫分子流行病学调查

为了掌握长春地区梅花鹿毕氏肠微孢子虫(Enterocytozoon bieneusi)的感染率及基因型种类的分布情况,探究其在人与动物之间传播的源由,本研究于2018年12月-2019年12月期间采集长春地区8个养殖场共计538份梅花鹿粪便样品进行毕氏肠微孢子虫的流行病学调查及基因型分析,经PCR鉴定,测序分析、序列拼接比对以及系统进化分析。试验结果表明,长春地区梅花鹿毕氏肠微孢子虫的感染率为17.84%(96/538),不同养殖场的感染率为0~47.96%;研究共发现14种基因型(BEB6、EbpC、Ⅰ、JLD-Ⅲ、JLD-Ⅸ、JLD-ⅩⅤ、JLD-ⅩⅥ、JLD-ⅩⅦ、JLD-ⅩⅧ、JLD-ⅩⅨ、JLD-ⅩⅩ、JLD-ⅩⅪ、JLD-ⅩⅫ和JLD-ⅩⅩⅢ),其中BEB6为优势基因型。应用多位点序列分型(MLST)方法,选择MS1、MS3、MS4以及MS7位点对毕氏肠微孢子虫阳性样本进行多态性和群体遗传结构分析,结果显示,在96份阳性样品中,共有16份阳性分离株在至少2个位点同时扩增成功,形成10个多位点基因型(multilocus genotypes,MLGs),相同的基因型之间存在不同亚型,表明长春地区梅花鹿毕氏肠微孢子虫具有丰富的遗传多样性。本研究应用分子生物学方法对长春地区梅花鹿毕氏肠微孢子虫的流行病情况及基因型进行分析,研究结果不仅为梅花鹿毕氏肠微孢子虫防控提供科学依据,也为防控毕氏微孢子虫在人和动物间的传播提供基础数据,对保障公共卫生与健康意义重大。     

First report of Enterocytozoon bieneusi infection on a pig farm in the Czech Republic

Enterocytozoon bieneusi infects humans and animals and can cause life-threatening diarrhea in immunocompromised people. The routes of transmission and its zoonotic potential are not fully understood. Pigs have been frequently reported to have E. bieneusi; therefore, we surveyed farm-raised pigs in the Czech Republic to determine its presence and genetic diversity. Spores were detected by microscopy in the faeces of 65 out of 79 examined animals (82%). A species-specific polymerase chain reaction (PCR) identified E. bieneusi in 94% of samples. Genotyping based on the ITS regions of the SSU rRNA gene identified that most pigs were infected with the species-specific genotype F, while two animals had the zoonotic genotype D and two had genotype Peru 9. This is the first report of E. bieneusi in swine in the Czech Republic, and demonstrated that most infections were with pig-specific genotypes. Nonetheless, swine may still play a role in the transmission of E. bieneusi to humans.     

云南省怒江州部分地区独龙牛毕氏肠微孢子虫感染情况及基因分型研究

为了解云南省怒江州独龙牛毕氏肠微孢子虫感染情况,从怒江州的鸠门当、古泉村、亚左洛村、茨开镇、独龙江乡分四个季度(春、夏、秋、冬)采集独龙牛粪便样本1 129份,采用分子生物学技术对其毕氏肠微孢子虫感染情况进行调查研究.结果发现,所调查地区独龙牛毕氏肠微孢子虫总感染率为1.68％(19/1 129);各地点之间感染率差异...     

Longitudinal identification of Enterocytozoon bieneusi in dairy calves on a farm in Southern Xinjiang,China

Enterocytozoon bieneusi is the most common species responsible for human and animals microsporidiasis. A total of 250 samples were collected weekly from 25 newborn dairy calves of a farm in Southern Xinjiang, China at one to ten weeks of age. Enterocytozoon bieneusi was identified and genotyped by nested PCR amplification and sequencing of internal transcribed spacer (ITS) region.The cumulative prevalence of E. bieneusi infection was 100% (25/25), and the average infection was 52.0% (130/250). The highest infection rate was recorded at six weeks of age (92.0%, 23/25), and no infection was observed at one and two weeks of age. Sequencing analysis showed nine E. bieneusi genotypes (J, EbpC, PigEBITS5, CHV4, CHC3, CS-9, KIN-1, CH5, and CAM5) were identified. The highest genetic polymorphism was observed at ten weeks of age. Genotype J was the predominant E. bieneusi genotype. Phylogenetic analysis clustered genotype J into Group 2 and other eight genotypes (EbpC, PigEBITS5, CHV4, CHC3, CS-9, KIN-1, CH5, and CAM5), detected in 22 (16.9%, 22/130) samples, into Group 1. Among the genotypes, EbpC, KIN-1, and J have been identified in humans. The highest E. bieneusi infection rate (57.9%, 124/214) was observed in fecal samples with formed feces with no diarrhea (p < 0.01), and high genetic polymorphism was observed in class I fecal samples. The presence of zoonotic E. bieneusi genotypes in dairy calves suggests the possibility of transmitting zoonotic infections to humans. It provides the basic data on dynamic change of E. bieneusi in calves.     

Cryptosporidium hominis Subtypes and Enterocytozoon bieneusi Genotypes in HIV‐Infected Persons in Ibadan,Nigeria

Cryptosporidium and Enterocytozoon are common opportunistic pathogens in HIV+ patients in developing countries, especially those do not have access to antiretroviral therapy. To determine the distribution of genotypes/subtypes of Cryptosporidium and Enterocytozoon bieneusi, faecal specimens were collected from 132 HIV+ persons attending a tertiary hospital in Ibadan, Nigeria. By polymerase chain reaction, eight and ten patients were identified as positive for Cryptosporidium spp. and E. bieneusi, respectively. Seven of the Cryptosporidium specimens were identified as C. hominis, while the remaining one as the new species C. viatorum recently identified in the United Kingdom. DNA sequencing of the 60‐kDa glycoprotein gene showed that the C. hominis belonged to three common subtype families: Ia (in three patients), Ib (in one patient) and Ie (in one patient). In contrast, DNA sequencing of the E. bieneusi internal transcribed spacer products showed the occurrence of genotypes associated with both humans (Peru 8 in one patient, Nig2 in two patients and a new genotype in one patient) and animals (D in one patient and Type IV in five patients). Low CD4+ cell count was identified as a risk factor for both cryptosporidiosis and microsporidiosis.     

Cryptosporidium, Giardia and Enterocytozoon bieneusi in cats from Bogota (Colombia) and genotyping of isolates

The prevalence of Cryptosporidium, Giardia, and Enterocytozoon bieneusi in cats from Bogota (Colombia) was determined from fecal specimens and scrapings of duodenal and ileal mucosa screened by PCR. All PCR-positive specimens were sequenced to determine the genotype(s) present. Of 46 cats, 6 (13%) were positive for Cryptosporidium, 5 (11%) were infected with C. felis and one (2%) with C. muris. Three (6.5%) cats were infected with Giardia duodenalis Assemblage F. Eight (17%) cats were infected with four genotypes of E. bieneusi: genotype D-like (9%), K (4%), Peru 10 (2%), and Peru 5 (2%). This is the first report on the presence of zoonotic species/genotypes of Cryptosporidium and E. bieneusi in cats in Colombia.     

Outbreak of tuberculosis caused by Mycobacterium caprae in a zoological garden

In the autumn of 2004, tuberculosis caused by Mycobacterium caprae occurred in a zoo in Slovenia. A dromedary camel (Camelus dromedarius) was killed after a history of progressive emaciation. Necropsy findings indicated disseminated tuberculosis, which was confirmed by cultivation of M. caprae. Consequently, a tuberculin skin test was performed in all epidemiologically linked animals and another dromedary camel and six bison (Bison bison) were positive and killed. Mycobacterium caprae was isolated from two bison while M. scrofulaceum and Mycobacterium spp. were found in two other bison, respectively. The second dromedary camel was found to be negative for mycobacteria under both microscopic and culture tests. The isolates were investigated with commercial identification kits, IS6110 PCR, IS6110 restriction fragment length polymorphism analysis, spoligotyping and mycobacterial interspersed repetitive units typing. Genotyping results revealed that the dromedary camel and the two bison were infected by the same M. caprae.     

Occurrence and molecular characterization of Cryptosporidium spp. and Enterocytozoon bieneusi in dairy cattle,beef cattle and water buffaloes in China

Cryptosporidium spp. and Enterocytozoon bieneusi are important protists in a wide range of vertebrate hosts, causing diarrheal diseases. Cattle are considered potential reservoirs of Cryptosporidium infection in humans, although their role in the transmission of E. bieneusi is not clear. In the present work, 793 fecal specimens from dairy cattle, native beef cattle, and water buffaloes on 11 farms in China were examined for the presence of Cryptosporidium spp. and E. bieneusi using nested PCR targeting the small subunit (SSU) rRNA gene of Cryptosporidium spp. and the internal transcribed spacer (ITS) of E. bieneusi. For Cryptosporidium, 144/446 (32.3%) dairy cattle, 44/166 (26.5%) beef cattle, and 43/181 (23.8%) water buffaloes were PCR-positive. Sequence analysis was successful for 213 of the 231 Cryptosporidium-positive isolates; among them 94 had Cryptosporidium andersoni, 61 had Cryptosporidium bovis, 54 had Cryptosporidium ryanae, 2 had a Cryptosporidium suis-like genotype, and 2 had mixed infections of C. bovis and C. ryanae. In dairy and beef cattle, C. andersoni and C. bovis were the most common species, whereas C. ryanae was the dominant species in water buffaloes. The latter species produced SSU rRNA sequences different between cattle and water buffaloes. For E. bieneusi, the infection rate of E. bieneusi in dairy cattle, beef cattle and water buffaloes was 4.9%, 5.4% and 2.2%, respectively. All 35 E. bieneusi-positive specimens were successfully sequenced, revealing the presence of four genotypes: three Group 2 genotypes previously reported in cattle as well as humans (I, J and BEB4) and one Group 1 genotype recently reported in yaks (CHN11). Genotypes I and J were the most common genotypes in dairy and beef cattle, while genotype CHN11 was the only genotype seen in water buffaloes. Thus, the distribution of Cryptosporidium spp. and E. bieneusi in water buffaloes might be different from in dairy and beef cattle in China. These findings indicate that some of the Cryptosporidium species and all four E. bieneusi genotypes identified in bovine animals in the study areas may have zoonotic potential.     

Prevalence and molecular characterization of Cryptosporidium spp. and Enterocytozoon bieneusi from large-scale cattle farms in Anhui Province, China

To investigate the prevalence of Cryptosporidium spp. and Enterocytozoon bieneusi from large-scale cattle farms in Anhui Province, 955 fecal samples were collected from 16 cattle farms from March to October 2018, which included six dairy farms (526), seven yellow cattle farms (323), and three water buffalo farms (106) in different regions of Anhui Province. PCR was conducted on all fecal samples using the 18S ribosomal RNA of Cryptosporidium spp. and internal transcribed spacer gene of E. bieneusi to detect these two pathogens, and the positive samples were sequenced and analyzed. The results showed that 23 (2.4%) and 40 (4.2%) out of the 955 samples were positive for Cryptosporidium spp. and E. bieneusi, respectively. There were 11 (2.1%), 10 (3.1%), and 2 (1.9%) positive samples of Cryptosporidium spp. and 16 (3.0%), 23 (7.1%), and 1 (0.9%) positive samples of E. bieneusi collected from dairy cattle, yellow cattle, and water buffalo, respectively, and no co-infection was identified in this study. All positive samples of Cryptosporidium spp. were C. andersoni with some variations. Ten E. bieneusi genotypes were obtained, including two known genotypes, J and CHN11, and eight new genotypes, named AHDC1 and AHYC1-7. The genotype CHN11 belonged to zoonotic Group 1, and the other nine genotypes belonged to Group 2, which is mainly documented in ruminants. These results indicated that Cryptosporidium spp. and E. bieneusi infections were present in large-scale cattle farms in Anhui Province. Therefore, attention should be paid to the development of containment strategies of these two pathogens in cattle.     

Upper respiratory tract infection caused by Cryptosporidium baileyi in three mixed-bred falcons (Falco rusticolus x Falco cherrug)

Three mixed-bred raptors (Falco rusticolus x Falco cherrug) from a German falcon breeder were presented with a history of respiratory distress. In one bird a laryngeal stridor was noted, and oral examination revealed an epiglottal swelling. In the other two birds, nasal discharge and sneezing were the main clinical symptoms. Nasal flushing samples and biopsies were collected for pathologic, bacteriologic, and parasitologic examination. Results confirmed a cryptosporidial infection. Polymerase chain reaction (PCR) and DNA analysis identified the causative agent to be Cryptosporidium baileyi. No cryptosporidia were detected in fecal samples, indicating the infection was confined to the respiratory system. Analysis of prey animals (pigeons, quail) failed to identify the source of infection. Treatment was initiated with paromomycin in all three birds, whereas in two birds an additional therapy with azithromycin was given. However, no clinical improvement was seen after several weeks of treatment, and the birds either died or were euthanatized. To the authors' knowledge, these are the first confirmed cases of disease caused by cryptosporidia in the order of Falconiformes.     

Outbreak of bovine clinical mastitis caused by Mycoplasma bovis in a North Italian herd

This report describes an outbreak of Mycoplasma bovis mastitis affecting 45 cows in a herd of 122 dairy cattle in Northern Italy. Clinically, the outbreak was characterized by agalactia, multiple swollen and painless quarters, high milk somatic cell count and unresponsiveness to conventional antibiotic therapy. M. bovis was isolated from the milk samples of all the 32 affected cows tested and from the mammary tissue of three affected cows that underwent necropsy. No other pathogens were isolated from these samples. Lesions in two of the necropsied cows were characterized by mild chronic suppurative mastitis and galactophoritis. The other necropsied cow showed a chronic necrosuppurative and pyogranulamaous galactophoritis, a condition not previously associated with M. bovis. M. bovis was detected immunohistochemically in the lumen of the affected mammary ducts suggesting that ascending infection via the teat canal was the likely route of transmission. No other intralesional pathogens were demonstrated microscopically.