Mutation scan screening of Echinococcus granulosus isolates of Indian origin

During the present investigation a total of forty Indian animal isolates were screened by single strand conformation polymorphism (SSCP) collected from sheep, goat, cattle and buffalo. The result of the study indicated that nuclear variants of Echinococcus granulosus were present in both small and large ruminants. SSCP phenotypes of AgB, intron of actin II and Hbx-2 have been deduced. Presence of nuclear variants due to mutation of E. granulosus has been discussed depending on hypotheses imparted earlier in literature. High polymophism of AgB demands further investigation because the gene is related with immune evasion and infectivity. This communication reports for the first time the comparative profile of Indian goat, sheep, cattle and buffalo isolates of E. granulosus complex.     

Molecular discrimination of sheep and cattle isolates of Echinococcus granulosus by SSCP and conventional PCR in Turkey

Cystic echinococcosis (CE), caused by hydatid cysts, is a widespread and hazardous disease in humans and animals worldwide. The aim of the current study was to investigate the genetic characteristics of sheep and cattle isolates of Echinococcus granulosus obtained from eastern Turkey using Single Stranded Conformation Polymorphism (SSCP) analysis and conventional PCR method. A total of 54 isolates collected from Erzurum and Elazig provinces of east-Turkey were examined. The 31 of these were obtained from liver of sheep while 23 cattle isolates (12 of liver and 11 of lung) were tested. After the total genomic DNA isolation 12S rRNA gene of all isolates were examined by PCR for the aim of genetic characterization by conventional PCR and mitochondrial CO1 gene for SSCP analysis. The 12S rRNA-PCR yielded 254 bp of amplification product with all samples analyzed. Thus, these samples were identified as G1-G3 cluster (E. granulosus sensu stricto). At least two major single stranded bands were resolved for G1-G3 cluster and G5 in SSCP analysis. While the resolution of more than two additional single stranded bands in SSCP indicated the existence of G7 genotype. The SSCP analysis was identified the G5 and G7 while failed to G1 and G3. The present SSCP analysis classified all 54 cyst isolates from sheep and cattle as E. granulosus sensu stricto (G1-G3 cluster). However, some sequenced samples for G1 and G3 showed the same band patterns by SSCP.     

Genotypic characterisation of Indian cattle, buffalo and sheep isolates of Echinococcus granulosus

Twelve isolates of Echinococcus granulosus, collected from domestic animals, including cattle, buffalo and sheep were analysed for DNA nucleotide sequence variation within mitochondrial cytochrome c oxidase I (coxI), NADH dehydrogenase subunit I (nadI) and internal transcribed spacer gene I (ITS1). After analysis of sequence information this was found that the fragment size of ITS1 of buffalo isolate was more in comparison to cattle and sheep isolates. Based on the nadI genotype this was found that Indian cattle, buffalo and sheep isolates could be grouped into E. granulosus sensu stricto. Based on coxI genotype two sheep isolates and one buffalo isolate were homologous to G2 genotype. Rests of the isolates were microvariants of G2 genotype. Presence of G2 genotype in buffalo is the first report of this genotype from this host.     

Variability in the Echinococcus granulosus cytochrome C oxidase 1 mitochondrial gene sequence from livestock in Turkey and a re-appraisal of the G1-3 genotype cluster

Investigations into the genetic strains of Echinococcus granulosus parasites occurring in sheep and cattle in Turkey were undertaken. A total of 112 hydatid cysts were investigated from sheep (100 isolates) derived from widely distributed sites within Turkey as well as from cattle (12 isolates) from the Turkish province of Kars. The parasite genotypes in these isolates were determined by DNA sequencing of part of the mitochondrial Cytochrome C oxidase 1 (cox1) gene. Haplotypes were identified which corresponded clearly to the previously described strain G1 in a total of 107 isolates, including 98 isolates from sheep and 9 isolates from cattle. Five isolates, including 2 sheep and 3 cattle, were determined to belong to the G3 genotype. Parasites of the G3 genotype were identified only in isolates derived from animals in the eastern regions of Turkey. While the majority of the isolates described here had haplotypes corresponding to the G1 genotype, none matched exactly the G1 sequence that was defined in previous studies. Analysis of all GenBank entries for E. granulosus cox1 sequences representing G1, G2 and G3 genotypes identified substantial microsequence variability. G1 and G3 could be distinguished as separate strains, however, the existence of G2 as a separate strain could not be supported. Rather, this can be regarded as a microsequence variation of G3.     

Molecular epidemiology of Echinococcosis from food producing animals in north India

Echinococcosis is an important medical, veterinary and economic concern in India. Ten cysts were randomly selected from each intermediate host species (cattle, buffalo, sheep, goat and pigs). Either the germinal layer (sterile cysts) or protoscoleces (fertile cysts) were collected for molecular characterization. A 434 base pair fragment of the mitochondrial cytochrome oxidase-1 gene was amplified using PCR from each isolate. Ten representative samples (2 from each intermediate host species) were sequenced in both the directions from which readable sequences were obtained from nine for phylogenetic analysis (NCBI, Blast). Phylogenetic analysis of cytochrome oxidase I gene revealed that seven (77.7%) isolates, from cattle (2), pigs (2), buffaloes (1) and goat (2) were clustered with the Indian Buffalo (G3) strain of Echinococcus granulosus, while two (22.2%) isolates from sheep were clustered with the sheep strain (G1) of E. granulosus. Phylogenetic analysis of the cytochrome oxidase-1 gene revealed that the buffalo strain (G3) and common sheep strain (G1) are cycling among livestock in north India and that these strains are highly adapted to cattle, buffalo, sheep, goats and pigs.     

Cystic echinococcosis in Algeria: cattle act as reservoirs of a sheep strain and may contribute to human contamination

In Algeria, cystic echinococcosis (CE) is a serious economic and public health problem. The common sheep/dog cycle is usually considered as the major source of human contamination. But to date the main strain of Echinococcus granulosus involved in the human contamination and the role of other hosts are still unknown. This paper reports an original work performed in northern Algeria combining field observations and molecular analysis. In a first step, examination of 6237 carcasses in slaughterhouses showed high infection and fertility rates in cattle and dromedaries. Then, in a second step, we used a molecular biology approach to identify the E. granulosus strain(s) involved. Forty-six samples from various origins were collected. They were analysed using comparison of PCR-amplified DNA sequences with one genomic (BG 1/3) and two mitochondrial (COI and NDI) targets. Results show the presence of a "sheep" strain of E. granulosus in North Algeria circulating between cattle and ovines and infectious to humans, whereas in South Algeria, a "camel" strain and a "sheep" strain were found to circulate in camels and in sheep, respectively. This study also reports an ambiguous genotype which resembled the "sheep" strain genotype (Gl) on the basis of the partial COI gene sequence, whereas on the basis of the partial NDI gene sequence, it was similar either to the "sheep" strain (Gl) or to the "camel" strain (G6). Besides its basic interest, our study confirms the role of other hosts (mainly cattle) in leading to transmission to humans and suggests that control measures should not only target sheep.     

Infection of humans and animals with Echinococcus granulosus (G1 and G3 strains) and E. ortleppi in Southern Brazil

The Rio Grande do Sul state, in Southern Brazil, is one of the foci of human cystic echinococcosis (CE). The sheep strain (G1) of Echinococcus granulosus and Echinococcus ortleppi (also known as cattle strain G5) have been reported before to infect livestock. However, up to the present, no molecular data are available on isolates of the E. granulosus complex from humans and dogs. The present study analyzed hydatid cysts from 6 CE patients and adult worms from 12 dogs. Sequencing of the mitochondrial cox1 and 12S rRNA genes detected the E. granulosus G1 genotype from four human cases, the G3 genotype (or buffalo strain) from one human case and E. ortleppi from another human case, respectively. Ten of the twelve dogs were found infected with the G1 genotype, and one dog each harbored worms of the G3 genotype and E. ortleppi. Obvious morphological differences were recognized between the G1 and E. ortleppi adult worms from dogs in this region. The buffalo strain (G3) is for the first time reported from South America.     

IS900-PCR-based detection and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep

Johne's disease is one of the main causes of economic losses in ruminants and a major health hazard in the developing and developed world. Up till now, many microbiological, serological and molecular methods have been tried for the detection of Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we attempt a PCR-based detection of IS900, distinct insertion sequences of MAP from the buffy coat of cattle (n=262) and sheep (n=78), and direct genotyping by single strand conformational polymorphism (SSCP). A total of 30 (11.45%) cattle and one sheep (1.28%) were positive for MAP-IS900. This IS900-based PCR detection proved highly specific, particularly when tested on other non-MAP strains. SSCP analysis grouped the MAP-IS900 into four distinct clusters based on different band patterns. Nucleotide sequence variability between MAPs detected from sheep (GenBank accession ) and cattle (GenBank accession -) was noticed in the study. Although, in recent years IS900-PCR-based detection of MAP from WBCs is being used in human, its use in animals is still limited. Our work not only supports its use in animals but also suggests further IS900-SSCP-based MAP-genotyping, coupled with DNA sequencing, as a promising tool for rapid and effective Johne's disease surveillance.     

Molecular evidence of ovine (G1) and camel (G6) strains of Echinococcus granulosus in Tunisia and putative role of cattle in human contamination

Three hundred and seventy-two cysts coming from 50 humans, 166 cattle, 153 sheep and 3 camels were collected in order to establish some epidemiological molecular information in Tunisia for the first time. The analysis by PCR-RFLP of ITS1 sequence showed that all the human, ovine and bovine cysts were due to the common sheep strain of Echinococcus granulosus. The sequencing of the CO1 gene of 37 isolates confirm the G1 genotype of this strain. For seven of these isolates, we found the mutation C56T which is present in the three principal intermediate hosts: human (three cysts), cattle (three cysts) and sheep (one cyst). With regard to the G1 genotype, we identified three other point mutations. The camel strain G6 is uniquely found in the three camels isolates and not in the other intermediate hosts analysed. The fertility of the bovine cyst represents 48% that means that this host is involved in a bovine-dog cycle and consequently represents a reservoir of sheep strain in Tunisia. Our results confirm the importance of the prophylaxis measures in order to disrupt the cycle of transmission sheep-dog in Tunisia. Nevertheless, the supervision of bovine infection should be reinforced because this intermediate host may constitute an important link with the human contamination.     

Allelic polymorphism in the ovine DQA1 gene

Variation in the ovine DQA1 gene was investigated by amplification of exon 2 using PCR, followed by single-strand conformational polymorphism (SSCP) analysis, cloning, and DNA sequencing. Fourteen novel SSCP patterns, representing 14 different sequences, were identified. Eight of these 14 sequences were identical to published DQA1 sequences from sheep, whereas the remaining six were novel but similar to the published DQA1 sequences from sheep and cattle. These six new sequences exhibited conserved region and variable region patterns similar to the published sheep DQA1 sequences, but were different than the published DQA2 sequences from sheep. All of these 14 putative sheep DQA1 sequences fulfilled the criteria used by the established bovine leukocyte antigens major histocompatibility complex nomenclature committee for assignment as new alleles. Comparison of the available DQA1 sequences from sheep and cattle revealed several clusters of ovine DQA1 sequences, and some sheep alleles were more similar to cattle alleles than other sheep alleles. The occurrence of trans-species polymorphism suggests the action of balancing selection at the DQA1 locus. Twenty-four percent of the nucleotide positions showed variation within exon 2, and this variation seems to have arisen largely by point mutation and gene conversion. The nonsynonymous and synonymous substitution rates were similar in both the putative antigen-binding site codons and the putative nonantigen-binding site codons. The extensive polymorphism reported in this article is consistent with polymorphism reported at the bovine DQA1 locus.     

Morphological and molecular characteristics of Echinococcus multilocularis and Echinococcus granulosus mixed infection in a dog from Xinjiang, China

The Xinjiang plateau of western China has been shown to have a high prevalence for human cystic echinococcosis (CE) caused by Echinococcus granulosus, and human alveolar echinococcosis (AE) caused by Echinococcus multilocularis. The domestic dog is suspected to be the primary definitive host for the transmission of both E. granulosus and E. multilocularis to humans in this locality. Seventeen of 30 stray dogs from Hejing County of Xinjiang were found positive for E. granulosus post mortem, and one double infection was suspected. Worm samples were collected, dyed by carmine, and observed microscopically. Carmine staining examination clearly revealed the differences in number of proglottids and appearance of uterine branches and lateral genital pore for those two species of Echinococcus. Furthermore, gene target DNA fragments were amplified for formal identification of the two parasite species, based on 12s rRNA mitochondrial gene. The PCR products were purified and sequenced. Compared with NCBI GenBank, the DNA sequences demonstrated 100% identity with E. granulosus (sheep strain, G1 genotype) and E. multilocularis.     

Detection of patent infections of Echinococcus granulosus ("sheep-strain", G1) in naturally infected dogs in Kosovo

A survey was carried out to assess the occurrence of canine echinococcosis in naturally infected dogs in Kosovo. Using the flotation-ovassay technique, taeniid eggs were found in 23 (7.5%) out of a total of 305 dogs. Eggs from other helminths were detected as well: hookworms 139 (45.5%), Trichuris sp. 87 (28.5%), Toxocara sp. 42 (13.7%), Toxascaris leonina 21 (6.8%) and Dipylidium caninum eight (2.6%). From 21 of the 305 samples (6.9%), taeniids eggs could be collected. Using PCR primers specific for Echinococcus granulosus ("sheep strain", G1), four of these samples (1.3%) resulted positive. The E. granulosus isolates originated from each one stray dog, hunting dog, sheepdog and pet dog. A semi-quantitative analysis showed low to moderate egg counts (2-10 per 1 g faeces) in dogs positive for E. granulosus ("sheep strain", G1) whereas specimens with high (11-20) or very high numbers (> 20) of taeniid eggs were negative in the E. granulosus PCR. Using specific primers for the detection of E. multilocularis, all samples containing taeniid eggs were negative. This is the first report on identification of E. granulosus in dogs from Kosovo where human cystic echinococcosis is a significant medical problem.     

基于线粒体12S基因对青海地区细粒棘球蚴种群遗传多态性研究

本研究旨在分析青海地区细粒棘球蚴的种群基因多态性,为细粒棘球蚴病的防控提供基础资料。对采自青海地区的42株细粒棘球蚴(33株采自绵羊肝脏,9株采自绵羊肺脏)进行了线粒体12S基因的全序列测序并构建了NJ系统发生树。结果显示:在本研究样品的线粒体12S基因序列中共检测出5种单倍型(即H1～H5),其中以单倍型H5为主(占32株),并且系统发生树分析支持这一结果。单倍型多样性(H)和核苷酸多样性(Pi)分别为0.418、0.000 66,与E.granulosus G1(AF297617)的12S基因序列的核苷酸相似性达到99.86%以上。采自青海地区的42株细粒棘球蚴均鉴定为E.granulosus sensu stricto(基因型G1-G3),在检测出的5种单倍型中,单倍型H1～H4是本地区特有的单倍型。     

Genetic variation within and between G1 and G3 genotypes of Echinococcus granulosus in Italy revealed by multilocus DNA sequencing

Numerous studies have provided evidence that Echinococcus granulosus exists as a complex of different strains, that differ in a wide variety of criteria that have an impact on the epidemiology, pathology and control of cystic hydatid disease (CHD) and, to date, 10 distinct genotypes (G1-G10) have been identified. In Italy, sequence analysis of the mitochondrial cox1 and nad1 genes showed the occurrence of the G1 genotype, the common sheep strain, the G3 genotype, the buffalo strain and of one isolate identified as G2 genotype, the Tasmanian sheep strain. In the present work, we have analysed E. granulosus strains in Italy, by genotyping a large sample of isolates and by checking out the genetic differentiation within and among the G1 and G3 genotypes using an additional mitochondrial gene as marker, the rrnS gene. Sequencing of the rrnS gene revealed a significant genetic differentiation between isolates identified as belonging to the G1 and G3 genotypes, with fixed nucleotide substitutions. This study provides further evidence of the occurrence of the E. granulosus G3 buffalo strain in Italy, a strain previously thought to be confined to the Indian region.     

Molecular update on cystic echinococcosis in cattle and water buffaloes of southern Italy

Cystic echinococcosis (CE)--caused by the larval stage (hydatid cyst) of the cestode Echinococcus granulosus--is one of the most widespread zoonoses of veterinary and medical importance. Molecular techniques have allowed the identification of 10 different genotypes (G1-G10) of the parasite. The present paper is an update regarding the E. granulosus genotypes infecting water buffaloes and cattle bred in the Campania region of southern Italy. The molecular study was performed on 30 hydatid cysts (11 from water buffaloes and 19 from cattle). Two different mitochondrial DNA genes, namely the cytochrome c oxidase subunits 1 and the 12S ribosomal DNA (12S rDNA) were used as genetic markers. Three different genotypes of E. granulosus were unequivocally identified, i.e. the G1 (common sheep), G2 (Tasmanian sheep) and G3 (buffalo) genotypes, as well as some G1 and G2 variants. It should be noted that the present study demonstrated for the first time: (i) the presence of the G2 genotype in water buffaloes from a Mediterranean area; and (ii) the fact that the analysed portion of the 12S rDNA gene can not discriminate between the G2 and G3 genotypes of E. granulosus. The finding of the G1, G2 and G3 genotypes in large ruminants from southern Italy is of epidemiological relevance and immediate public health importance because of their recognized infectivity in humans.     

Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species

The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).     

Study on Length Polymorphism of KAP1 Family Genes in Yak (Bos gurnniens)

This study was aimed to understand the characteristics of length polymorphism with repeat sequence of keratin associated protein 1 (KAP1) family genes in yak. KAP1 family genes of yak and cattle were sequenced, and compared with sheep KAP1 family gene sequences. The results showed that cattle KAP1 family genes were located in chromosome 19, according to location of sheep KAP1 family genes in the chromosome and similarity with cattle KAP1 family genes, renaming the cattle KAP1 family (according to the gene location of chromosome) B2D, B2A, KAP1-1 and B2C genes into KAP1-4, KAP1-1, KAP1-2 and KAP1-3 gene, respectively. KAP1 family genes in the 3'and 5' flank were highly conserved, the difference between family genes mainly in the the repeat sequence region, which yak KAP1 to KAP4 genes were found 30 bp length polymorphism. There were B(CCQTS)A₁(CCQPT) repeat sequence and a new repeat sequence C(SIQTS). The results indicated that the repeat sequence was the key of the polymorphism of KAP1 family genes, which might be relate to combination with keratin protein.     

牦牛KAP1家族基因长度多态研究

试验旨在研究牦牛角蛋白关联蛋白1（keratin associated protein 1,KAP1）家族基因长度多态与重复序列特点。研究对牦牛和黄牛KAP1家族基因进行测序,并与绵羊已知序列进行比较分析。结果发现,牛KAP1家族位于19号染色体,根据绵羊KAP1家族基因在染色体上的位置与相似性,重新命名了牛KAP1家族基因B2D、B2A、KAP1-1和B2C为KAP1-4、KAP1-1、KAP1-2、KAP1-3（按照染色体上的基因顺序）。KAP1家族基因之间在3'和5'端区域高度保守,中间有重复序列长度差异,其中牦牛KAP1-KAP4基因发现有30 bp的长度多态。研究其蛋白序列发现5个氨基酸为基序的重复序列B（CCQTS）A₁（CCQPT）,以及一个新的重复序列C（SIQTS）。本研究结果说明重复序列是KAP1家族基因间和基因内的主要差异区域,这可能与其角蛋白结合螺旋数相关。     

Orf virus circulation in cattle in Turkey

Orf virus (ORFV) causes contagious skin disease that mainly affects sheep and goats with zoonotic potential. However, there is not enough information about the association between ORFV and occurrence of skin disease in cattle. The present study describes outbreaks of ORFV infection in cattle in different provinces that are located in the Aegean, Central Anatolian and Mediterranean regions of Turkey. During the months of June and August 2017, vesicular fluid and scab samples were collected from cattle which had proliferative skin lesions. First, presence of lumpy skin disease virus (LSDV) and bovine herpesvirus 2 (BoHV-2, known as the causative agent of pseudo-lumpy skin disease) were investigated by real time PCR and PCR, respectively. Then, samples tested for the presence of parapoxviruses by PCR using primers specific to major envelope protein gene (B2L). Parapoxvirus DNA was detected in investigated samples whereas LSDV and BoHV-2 DNA were not detected. The analysis of the B2L gene sequences revealed that cattle were infected with ORFV. The isolates in the present study shared 100% sequence identity at the nucleotide and amino acid level when compared with previously characterised Turkish field ORFV isolates from goats in 2016. Results of the study show unusual infection of cattle with ORFV, and suggest that ORFV jumps the host species barrier from goats to cattle.     

DNA fingerprinting of Australian isolates of Mycobacterium avium subsp paratuberculosis using IS900 RFLP

OBJECTIVES: To evaluate additional restriction enzymes for IS900 RFLP of Mycobacterium avium subsp paratuberculosis and examine the genetic diversity among Australian isolates for epidemiological studies of Johne's disease. DESIGN AND PROCEDURE: Seventy-one isolates of M paratuberculosis from cattle, sheep, goat, alpaca and rhinoceros in six Australian States and the Northern Territory, reference strains and reference DNA from previously characterised strains were tested for genetic variation. Bst EII, Pvu II and Pst I restriction enzymes were used, and four others (Bam HI, Alu I, Xho I and Dra I) were assessed for their ability to detect polymorphisms. Multiple isolates from some animals were tested. RESULTS: Bam HI, was the most effective enzyme for identifying polymorphisms (12 types), followed by Bst EII (11 types). Both Pvu II and Pst I were relatively ineffectual. Fifteen different types were identified, 12 in clinical isolates. Most isolates were cattle (C) strains and fell into the C1 (n = 28) and C3 (n = 32) groupings. All isolates from alpaca were type C1, and bovine isolates were commonly C1 (n = 15) or C3 (n = 28). All of the sheep were infected with sheep (S) strains; no S strains were identified in cattle. Two of six isolates from one animal had single band differences. CONCLUSION: The epidemiological features of M paratuberculosis in Australia are similar to those reported in New Zealand, where cattle and sheep are commonly infected with different strains. However, because of the lack of polymorphism identified within the major groups, it is unlikely that DNA fingerprinting will have a significant role in epidemiological studies of Johne's disease, unless an unusual strain in being studied.