First report of Cryptosporidium deer-like genotype in Kenyan cattle

The objective of the study was to identify Cryptosporidium genotypes from feces collected from urban and peri-urban dairy cattle in Nairobi, Kenya, in order to determine their zoonotic potential. DNA was extracted from 34 samples that were diagnosed positive by the modified Ziehl-Neelsen technique. Two Cryptosporidium isolates examined at the 18S rRNA locus were identified as the deer-like genotype by DNA sequencing. As public health officials are facing the difficult decision whether to allow urban livestock production because of its economic benefits and a livelihood asset to the urban communities, or to ban it for its public health risks, the finding of non-zoonotic genotypes in a smallholder dairy system has significant public health as well as economic implications that merit further investigation.     

First report of Cryptosporidium deer-like genotype in Malaysian cattle

Fifty faecal samples from diarrheic calves between 1 and 6 months old were collected per rectum from 5 farms around Petaling District in Selangor, Malaysia for Cryptosporidium species detection and genotyping investigation. Oocysts were purified using sedimentation and gradient centrifugation, then examined by immunofluorescence assay (IFAT). Genomic DNA was extracted from all samples and nested PCR was performed to amplify the SSU rRNA gene. Eighteen samples (36%) were positive for Cryptosporidium species by PCR. The sequence and phylogenetic analysis of 14 isolates indicated that Cryptosporidium parvum was most common (11 isolates) followed by Cryptosporidium deer-like genotype (3 isolates). The present work reports the first data on Cryptosporidium genotyping from cattle in Malaysia.     

High prevalence of Cryptosporidium bovis and the deer-like genotype in calves compared to mature cows in beef cow-calf operations

Recent studies have identified the novel, host adapted Cryptosporidium bovis and the deer-like genotype in dairy cattle from farms in the United States, China, India and Europe. This novel species and genotype appear to be more prevalent in older, post-weaned dairy cattle than previously thought. However, little information is available on their prevalence in beef cow-calf operations. In the present study, we determined the prevalence of Cryptosporidium species in 98 calves (6-8 months old) and 114 cows (>2 years old) in seven beef cow-calf herds in western North Dakota. DNA was extracted from fecal samples and Cryptosporidium spp. were identified by amplification of the 18S rRNA gene followed by sequencing or RFLP analysis. All seven herds tested positive for Cryptosporidium. Overall, 43/212 (20.3%) animals were positive. Only five of these positives were from cows. C. bovis, the deer-like genotype and C. andersoni were identified in 9.4, 6.6 and 1.4% of animals sampled, respectively. C. parvum was not identified in any of the positive samples. C. bovis, the deer-like genotype and C. andersoni were detected in 6/7, 5/7 and 2/7 herds, respectively. C. bovis and the deer-like genotype were primarily detected in calves, while C. andersoni was only detected in cows. Six isolates could not be typed. These results show a relatively high prevalence of C. bovis and the deer-like genotype in 6-8-month-old beef calves compared to cows older than 2 years in the seven herds studied.     

Age, geographic, and temporal distribution of fecal shedding of Cryptosporidium parvum oocysts in cow-calf herds

OBJECTIVE: To evaluate fecal shedding of Cryptosporidium parvum from California cow-calf herds with respect to age, geographic region, temporal effects, and association with watery feces. ANIMALS: Cows and calves from 38 beef cow-calf operations. PROCEDURE: Fecal specimens were collected and examined for C parvum oocysts, using immunofluorescent microscopy. Associations between age, geographic region, month of collection, watery feces, and likelihood of shedding C parvum were evaluated. RESULTS: 3.9% of cattle were shedding C parvum oocysts. Prevalence of shedding among calves ranged from 0 to 13%, and was 0.6% among cattle > or = 12 months old. The odds of shedding C parvum among 2-month-old calves were 41 times greater than among cattle > 4 months old. The odds of shedding C parvum among cattle tested in May were 8.7 times greater than among cattle tested during June, July, or August. The odds of infected individuals having watery feces were 3 to 4 times greater than for noninfected individuals, but the etiologic fraction was only 8 to 9%. CONCLUSIONS AND CLINICAL RELEVANCE: Substantial fecal shedding of C parvum by cow-calf herds was limited to calves 1 to 4 months old, with low prevalence detected in older animals. Risk of contamination of watersheds with C parvum was limited to those periods when young calves were in the herd. Although the odds of having watery feces were greater for animals infected with C parvum than for noninfected animals, the low etiologic fraction suggests that most calves with watery feces were not infected with C parvum.     

猪源隐孢子虫卵囊的分离及基因型鉴定

应用RT-PCR方法对南京、上海和合肥猪源隐孢子虫卵囊SSU rRNA部分序列进行扩增,产物测序后提交GenBank,收录号为DQ855266、DQ855267;用BLAST和DNAStar软件与GenBank参考序列进行比较,分析其同源性,绘制系统发育进化树,结合卵囊形态学观察和对小鼠、大鼠、兔、山羊和鸡的传染性试验确定隐孢子虫种类或基因型。结果表明,3地区猪源隐孢子虫分离株与微小隐孢子虫(C.parvum)同源性达94%~100%,与C.parvummouse型有99.8%~100%的同源性,并处于进化树的同一分支。因此,3地区猪源隐孢子虫是C.parvummouse型,提示猪和鼠之间存在交叉传播的可能。     

The prevalence of Cryptosporidium, and identification of the Cryptosporidium horse genotype in foals in New York State

To date, little is known about the prevalence, genotypes and zoonotic potential of Cryptosporidium spp. affecting horses, especially in North America. A cross-sectional study was conducted in New York, USA between February 25th and May 1st 2009. Fecal samples were collected from three hundred and forty nine 1-10-week-old foals and their dams on 14 different broodmare farms. All fecal samples were screened for Cryptosporidium spp. using a direct immunofluorescence assay (DFA). DNA extraction and PCR-RFLP analysis of the small-subunit (SSU) rRNA gene were performed on all the foal samples. PCR-positive samples were subtyped by DNA sequencing of the 60-kDa glycoprotein (gp60) gene. On DFA, 13/175 (7.4%) foal samples and 3/174 (1.7%) mare samples were designated positive for Cryptosporidium spp., whereas on SSU rRNA-based PCR, 9/175 (5.1%) foal samples were positive. Cryptosporidium PCR-positive foals were significantly older (13-40 days, median age of 28 days) compared with negative foals (4-67 days, median 18 days, p=0.02). The number of foals with diarrhea or soft feces was not significantly different between positive and negative foals (p=0.09). PCR-RFLP analysis of the SSU rRNA gene and DNA sequencing of the gp60 gene identified the parasite as subtype VIaA14G2 of the horse genotype. This is the first report of a group of foals affected with the Cryptosporidium horse genotype, which has recently been detected in humans. As other contemporary molecular studies have identified C. parvum in foals, it seems that equine cryptosporidiosis should be considered a zoonosis.     

New view on the age-specificity of pig Cryptosporidium by species-specific primers for distinguishing Cryptosporidium suis and Cryptosporidium pig genotype II

Two species of Cryptosporidium are commonly identified in pigs: Cryptosporidium suis and Cryptosporidium pig genotype II. Detection of Cryptosporidium spp. is routinely based on molecular methods such as polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) or gene sequencing. However, most of these methods are hampered by low sensitivity to mixed infections. As a solution of this problem, novel species-specific primers were designed and tested in the present study. Sensitivity of our primers was identical to genus-specific primers, but more (1:48) mixed infections were detected using these species-specific primers on 477 DNA samples originating from naturally infected pigs of different age categories. Our results show differences in age-dependent susceptibility of pigs to the infection. Whereas C. suis was found in all tested categories of pigs (1-12 week of age and sows), Cryptosporidium pig genotype II was recorded only in animals older than 6 week of age. Usage of species-specific primers could help to better the understanding of epidemiology of pig specific Cryptosporidium spp. and its occurrence, which, on the basis of our results, is underestimated.     

Factors associated with shedding of Cryptosporidium parvum versus Cryptosporidium bovis among dairy cattle in New York State

OBJECTIVE: To isolate and speciate Cryptosporidium DNA from fecal samples obtained from dairy cattle in New York State and identify factors associated with whether cattle were shedding Cryptosporidium parvum versus Cryptosporidium bovis. DESIGN: Cross-sectional study. SAMPLE POPULATION: 115 fecal samples positive for DNA coding for the Cryptosporidium 18S rRNA gene from dairy cattle in New York State. PROCEDURES: A PCR assay was used to amplify DNA from fecal samples; amplification products were submitted for bidirectional DNA sequencing. Logistic regression was used to test for associations between various host factors and Cryptosporidium spp. RESULTS: 70 of the 115 (61%) fecal samples were found to have C parvum DNA, 42 (37%) were determined to have C bovis DNA, and 3 (3%) were found to have C parvum deer-type DNA. The presence of diarrhea at the time of fecal sample collection, oocyst count, and breed were associated with whether cattle were infected with C parvum or C bovis, with animals more likely to be infected with C parvum if they had diarrhea, had a high oocyst count, or were Holsteins. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that C parvum and C bovis can be isolated from dairy cattle in New York State and that various factors affect whether cattle infected with Cryptosporidium spp are infected with C parvum or C bovis. Findings also lend credence to the theory that C bovis may be more host adapted and thus less pathogenic to dairy cattle than C parvum.     

Molecular and morphologic characterization of a Cryptosporidium genotype identified in lemurs

This study reports the molecular and morphologic characterization of a Cryptosporidium sp., identified in stools of captive lemurs Propithecus verreauxi coquereli. Stool samples were collected from seven animals (n=7) presenting episodes of diarrhea. Bright-field light microscopy of stool smears stained with modified acid-fast technique revealed the presence of Cryptosporidium sp. oocysts in four of the stool samples analyzed. All microscopically positive samples were confirmed by PCR using primers designed to amplify DNA fragments from two independent loci, i.e. the Cryptosporidium oocyst wall protein (COWP) gene and the small subunit ribosomal RNA (ssrRNA) gene. Phylogenetic analysis based on the full-length ssrRNA gene placed this isolate within a clade that contains all currently known C. parvum species/genotypes, closely related to the C. parvum pig genotype. Comparison with partial ssrRNA sequences available in the GenBank revealed 100% sequence identity with the genotype previously identified in Canadian patients. This finding was confirmed further by comparison of the COWP gene partial sequences.     

Detection of a novel genotype of Cryptosporidium in Antarctic pinnipeds

A study was conducted to investigate the presence of Cryptosporidium and Giardia in Antarctic marine mammals. A total of 270 faecal samples from different species of pinnipeds from different locations in the South Shetland Islands and Antarctic Peninsula were analysed by immunofluorescence microscopy and PCR. Cryptosporidium was detected by PCR in three samples from Southern elephant seals (Mirounga leonina) and 2 Weddell seals (Leptonychotes weddellii). However, no oocysts were observed in any of the samples by immunofluorescence microscopy. Molecular characterisation of the isolates, using the 18S rDNA, the HSP70 and the COWP loci, revealed the presence of a Cryptosporidium sp., previously reported from an Antarctic Southern elephant seal, in the elephant seals and a novel genotype in Weddell seals. Giardia could not be detected in any of the samples analysed.     

Genotyping of Mycobacterium bovis by geographic location within Mexico

The spacer oligonucleotide typing (spoligotyping) method was used to differentiate 62 Mycobacterium bovis isolates obtained from tissues with macroscopic lesions typical of tuberculosis in dairy cattle from different regions of Mexico. Our purpose was to see if a strain from one region was genetically different from those of other regions (with the long-term aim of doing molecular trace back of isolates obtained in the laboratory). Results from the genetic analysis indicate that M. bovis isolates cannot be grouped by geographic location due to a wide range of genetic types involved in dairy cattle infections. Isolates even from the same herd showed different spoligotypes but some isolates from different region had similar genetic patterns. Genetic typing without epidemiologic information does not seem to be a plausible method to trace back animals to source of origin to detect and eliminate sources of infection.     

牛分枝杆菌Spoligotyping和VNTR—MIRU的基因分型研究

为了探讨间隔区寡核苷酸分型(Spoligotyping)和数目可变串联重复序列·结核分枝杆菌散在分布重复单元(VNTR-MIRU)基因分型法用于国内牛分枝杆菌的基因分型研究,初步了解我国牛分枝杆茵基因型种类、分布以及2种方法在我国应用的可行性.本研究收集东北、西北、华北、华南4个地区分离的10株牛分枝杆菌分离株,分别采用Spoligotyping和VNTR-MIRU对这些分离株进行基因分型,比较两种方法的分型效果,评价其在牛分枝杆茵基因分型中的应用.结果表明,使用Spoligotyping方法,10株牛分枝杆菌呈现出4种基因型,使用VNTR-MIRU方法,10株分离株也呈现4种基因型;当两种方法联合使用时,可以分为5种基因型,其中的2个茵株具有独特的基因型,显示来自不同地区的牛分枝杆菌存在主要的流行型为BCG家族.将Spoligotyping和VNTR-MIRU联合使用,可有效提高牛结核病的分子流行病学调查和病原学的监测效果.     

A rare Cryptosporidium parvum genotype associated with infection of lambs and zoonotic transmission in Italy

An outbreak of cryptosporidiosis occurred in a mixed sheep/cattle farm of Central Italy in October 2011. A total of 450 ovines (250 sheep and 200 lambs) and 140 bovines (130 cows and 10 calves) were housed in two separated units, at the time of the outbreak. About half of the lambs had diarrhea due to Cryptosporidium sp. with a mortality rate of 80%; calves were not infected. Genomic DNA was extracted from an archived slide and from fecal specimens, and the parasite was identified as Cryptosporidium parvum by PCR and sequence analysis at the CpA135 gene. Genotyping at the GP60 gene showed the presence of a very rare genotype, IIaA20G2R1. Shortly after the outbreak was identified, the son of the farm's owner, aged 18 months, experienced an acute gastroenteritis and was hospitalized due to recurrent episodes of diarrhea, fever, vomiting and lack of appetite. The feces tested negative for bacteria and viruses, whereas cryptosporidiosis was diagnosed by microscopy and an immunochromatographic test. Molecular typing identified the C. parvum genotype IIaA20G2R1 in the feces of the child. This is the first case of transmission of cryptosporidiosis in Italy involving lambs as source of oocysts infectious to humans.     

Development of a tandem repeat-based multilocus typing system distinguishing Babesia bovis geographic isolates

Mini- and microsatellite sequences have proven to be excellent tools for the differentiation of strains and populations in several protozoan parasites due to their high variability. In the present work we have searched the genome of the tick-transmitted bovine hemoprotozoon Babesia bovis for tandem repeats (TRs) that could be useful for a multilocus typing system. Hundred and nineteen sequences were shortlisted and tested in five common B. bovis reference isolates originating from distinct geographic locations of North and South America: Texas, USA (T2Bo), Mexico (RAD and Mo7), and Santa Fe and Salta, Argentina (R1A and S2P, respectively). Satellite sequences were PCR-amplified using specific primers, separated by polyacrylamide gel electrophoresis, visualized by silver staining and sized. Fourteen TR sequences could be reliably amplified in all isolates and displayed length polymorphism. All primers used were specific for B. bovis and did not amplify genomic DNA from the bovine host or from Babesia bigemina, the principal co-infecting bovine parasite in the Americas, allowing their future use in field surveys. The 14 satellite markers identified are distributed throughout the four chromosomes of B. bovis as follows: chromosome 1 (n = 3), chromosome 2 (n = 2), chromosome 3 (n = 5), and chromosome 4 (n = 4). Within the five B. bovis isolates we identified nine satellite marker loci with two alleles, three with three alleles, one with four and another with five alleles. In comparison to Theileria parva, a bovine hemoprotozoan that pertains to the same piroplasmida order and owns a genome of similar size, the number of polymorphic TRs and the average number of alleles per TR locus seem to be significantly reduced in the B. bovis genome. Furthermore, the ratio of micro- to minisatellites in both B. bovis and T. parva is considerably lower than in other eukaryotes, as confirmed by bioinformatic analysis. The multilocus genotype of the five B. bovis isolates was assessed and the genetic distance between each other determined followed by cluster analysis based on neighbor joining. The resulting phenogram showed that B. bovis isolates segregated into three clusters according to their geographic origin. The presented marker system is suitable to explore various parameters of B. bovis populations such as genetic diversity, infection dynamics and their structure under different epidemiological situations, which are of crucial importance for improved control strategies.