Occurrence and molecular characterization of Acanthamoeba, Naegleria fowleri and Blastocystis in water samples from various sources in Egypt.

PCR-testing coupled to isolate sequencing was conducted to detect prevalence and various genotypes/subtypes of 3 neglected waterborne protists (Acanthamoeba, Naegleria fowleri and Blastocystis) in water samples from various sources in Dakahlia governorate, Egypt. Out of 62 protozoan-suspected samples by microscopy, Acanthamoeba was molecularly confirmed in 24 (38.7%) samples from various sources including tap water. Twenty Acanthamoeba isolates were successfully-sequenced; 18 were designated as the genotype T3 and 2 as T4. Naegleria spp. were detected in 6 (9.6%) samples from the Nile, of them 2 (3.2%) were identified as N. fowleri. Blastocystis spp. were found in 4 (6.4%) samples from waste water and ground water. Blastocystis subtype 2 was found in a sample from waste water, which may reflect human infection with this subtype and constitutes a public health hazard because waste water is occasionally discharged in the Nile with minimal treatments. Findings of the present study were analyzed in combination with those of earlier surveys from the other Egyptian governorates to evaluate the whole situation of the 3 protists in water from Egypt. Results of this analysis showed that Acanthamoeba had a high mean prevalence (43.03%) throughout Egypt, with insignificant variations among various water sources. Various Acanthamoeba genotypes were detected, and the highly pathogenic T4 was the most significantly identified type. A common T4 haplotype circulated in water from Egypt and 3 other countries (Tanzania, Rwanda, Uganda) located on the Nile basin, and included isolates from keratitis-infected patients, which confirms the potential role of water in the epidemiology of Acanthamoeba keratitis infecting humans in these countries. The estimated mean prevalence for Naegleria spp. was 23.79%, being the highest in the Nile water. In the present study, occurrence of 3 potentially pathogenic protists has been confirmed in water from Egypt, which should alert the authorities to revise the procedures for controlling these pathogens in water.

Molecular identification of different Trichostrongylus species infecting sheep and goats from Dakahlia governorate, Egypt.

Nematodes of genus Trichostrongylus can cause remarkable economic losses in the small ruminant`s industry and some species have a zoonotic potential. Since the most common source for human infection is the infected animals, accurate identification of different Trichostrongylus species in animals would reflect the species that can infect humans from the same area. The objective of the present study is to identify common Trichostrongylus species infecting small ruminants in Dakahlia governorate, Egypt using molecular-based techniques. Fecal samples from 340 sheep and 115 goats from rural areas in 3 cities of Dakahlia governorate and 2 cities at its borders were collected, and the strongyle-type eggs were detected in 33.2% and 14.7% of sheep and goats, respectively. PCR amplification of the internal transcribed spacer of the ribosomal DNA (ITS2 rDNA) for 3 Trichostrongylus spp.; T. axei, T. colubriformis and T. vitrinus was conducted for eggs harvested from 25 sheep and 16 goat samples. Two species were detected; T. axei (in 16 sheep and 14 goats) and T. colubriformis (in 2 sheep but no goats). This is the first report of T. colubriformis in sheep from Dakahlia governorate, where this species was reported earlier from humans. No T. vitrinus was detected in any tested sample of sheep or goats. Purified PCR products of T. axei isolates were successfully sequenced and revealed 3 haplotypes; 2 from sheep and 1 from goats, and the isolates are related genetically to T. axei isolates from camels in Egypt. Phylogenetic analysis of the Genbank-retrieved ITS2-amplified T. axei isolates worldwide suggested the existence of genetic variants. Earlier reports on the identified Trichostrongylus spp. in different animals from Egypt as well as African and Arabian countries are tabulated.

Combined Molecular and Lectin Binding Assays to Identify Different Trichostrongyle Eggs in Feces of Sheep and Goats from Egypt.

BACKGROUND: Trichostrongyles are common causes of parasitic gastroenteritis in sheep and goats worldwide. Accurate identification of these nematodes to the genus and/or species level is important for therapy selection and control strategies. In the present study, molecular and egg-lectin binding approaches were employed to identify the most economically important trichostrongyles circulating in sheep and goat herds from six districts in Dakahlia governorate, Egypt. MATERIALS: Fecal samples from 653 and 205 goats reared within 17 herds were collected and tested for the trichostrongyle eggs using the modified Wisconsin sucrose flotation method. For identification of the trichostrongyle(s) present, eggs from 75 (63 sheep and 12 goats) samples which had high egg count (EPG) and pooled eggs (n = 19 pools, 15 sheep and 4 goats) from samples with moderate or low EPGs were examined. Molecular examination was conducted amplifying the ITS2 region of the rDNA for six different trichostrongyles in individual PCR reactions. For egg-lectin bindings, 4 fluorescently-labeled specific lectins were used; peanut agglutinin (PNA) for Haemonchus contortus, Aleuria aurantia agglutinin (AAL) for Trichostrongylus species, Lens culinaris agglutinin (LCA) for Teladorsagia circumcnicta and Lotus tetragonolobus lectin (LTL) for Cooperia species. RESULTS: Fourteen (82.3%) herds were found infected, of which trichostrongyle eggs were detected in fecal samples of 26.5% (173/653) of sheep and 10.2% (21/205) of goats. Results of the PCR and lectin bindings were compatible and 4 trichostrongyles were detected: H. contortus, T. circumcincta, Trichostrongylus axei and Trichostrongylus colubriformis. Haemonchus contortus eggs were found in all the infected herds, and as the single species in 21 and 5 of sheep and goat samples, respectively. Lectin stained smears demonstrated the dominance of H. contortus eggs over eggs of the other detected trichostrongyles. Eleven herds were found infected with T. axei as the second most prevalent trichostrongyle; however, few AAL-stained eggs were noticed in the positive samples. Mixed infections were frequently detected as H. contortus-T. axei combination. Infections with T. circumcincta were noted in sheep samples from two herds, but not in any sample from the goats. No Ostertagia leptospicularis, Cooperia curticei or Nematodirus species were noted among the tested samples. CONCLUSIONS: This is the first molecular and lectin binding survey to determine the species composition of trichostrongyles infecting sheep and goats from Egypt. Haemonchus contortus plays the principal role in small ruminant trichostrongylosis in Egypt. Egg-lectin staining shows promise for future for its application in routine diagnosis as a rapid and simple technique. Findings of the earlier reports from Egypt are tabulated and reviewed.

Prevalence of Eimeria species in sheep (Ovis aries) from Dakahlia governorate, Egypt.

Feces from 184 sheep from Dakahlia governorate, Egypt were tested for Eimeria species oocysts by using the standard floatation technique; oocysts were detected in 126 (68.4%). The prevalence was significantly higher in young sheep than adults. Eleven Eimeria species were identified: Eimeria ahsata, Eimeria bakuensis, Eimeria crandallis, Eimeria faurei, Eimeria granulosa, Eimeria intricata, Eimeria marsica, Eimeria ovinoidalis, Eimeria pallida, Eimeria parva and Eimeria webybridgensis. Oocysts of the most pathogenic ovine species, E. ovinoidalis, were detected in 27 (14.6%) sheep. This is the first report of E. webybridgensis in sheep from Egypt, possibly due to close similarity of their oocysts to those of E. crandallis which stated in the earlier reports. Worldwide reports on epidemiology of Eimeria spp. infections in sheep are tabulated.

First Evidence of Teladorsagia circumcincta Infection in Sheep from Egypt.

Trichostrongylid nematodes are a common cause of gastroenteritis in sheep. Despite its worldwide distribution, Teladorsagia circumcincta has not been included in reports listing the various trichostrongyles infecting sheep from Egypt. Herein, we describe the presence of 2 T. circumcincta haplotypes infecting small ruminants from Egypt. For this study, fresh fecal samples were collected from 340 sheep and 115 goats reared at 5 districts in Dakahlia governorate and its surroundings, Egypt. Trichostrongyle eggs were harvested from the samples, and then subjected to DNA isolation and analysis. Polymerase chain reaction (PCR) amplification was carried out for the second internal transcribed spacer of ribosomal DNA (ITS2 rDNA). Purified PCR products of T. circumcincta were sequenced, and the revealed sequences were subjected to the nucleotide and phylogenetic analysis. A relatively high prevalence of trichostrongyles eggs was identified in sheep (33.2%) and a lower prevalence was found in goats (14.7%). Molecular analysis revealed, for the first time, 2 sheep herds from Egypt that were infected with T. circumcincta. Both infected herds were raised by the Bedouins in rural areas of El Mahalla El Kubra city. No T. circumcincta infections were found in any of the goats. Nucleotide sequence analysis revealed 2 haplotypes (Te1 and Te2) from 7 successfully sequenced samples (5 from the first and 2 from the second herd). Te1 was the major haplotype in both herds, and Te2 was retrieved from a single sample. Phylogenetic analysis displayed that the Te1 haplotype clustered with one from Cyprus, which might have been introduced to Egypt via goats imported from Cyprus due to a program to improve meat and milk production in Egypt. The present results could be beneficial in understanding the epidemiology of T. circumcincta and other trichostrongyles in Egypt, and have implementations in the effective control strategies used in this region.

Molecular screening approach to identify protozoan and trichostrongylid parasites infecting one-humped camels (Camelus dromedarius).

Little is known about the diversity of many parasites infecting camels, with most relying on morphological parameters. DNA extracted from different tissues (n = 90) and fecal samples (n = 101) from dromedary camels (Camelus dromedarius) in Egypt were screened for multiple parasites using different molecular markers. Screening of tissue samples (heart) for Toxoplasma gondii and Sarcocystis spp. was performed using B1 and 18S rRNA gene markers, respectively. T. gondii was further genotyped using multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP). Sarcocystis was analyzed using PCR-RFLP characterization (XbaI and MboI restriction enzymes). A taxonomically challenging but important group of nematodes (Trichostrongylidae family) were screened using the ITS-2 ribosomal DNA (rDNA) species-specific markers. Furthermore, nested PCR was used for the detection of Cryptosporidium spp. (SSU rRNA gene) and positive samples were genotyped after RFLP (SspI and VspI) and sequencing. Cryptosporidium parvum isolates were subtyped by sequence analysis of the 60-kDa glycoprotein gene. This study revealed that many parasites infect the investigated camels, including T. gondii (1.1%), Sarcocystis spp. (64.4%), Cryptosporidium spp. (5.9%) and Trichostrongylidae nematodes (22.7%). The species contribution for nematodes was as follows: Haemonchus spp. (95.6%), Trichostrongylus axei (26%), Trichostrongylus colubriformis (65.2%) and Cooperia oncophora (60.8%). Mn-PCR-RFLP typing for Toxoplasma was only successful for three markers: 5'-SAG2 (type II), 3'-SAG2 (type II) and alt. SAG2 (type II). PCR-RFLP using XbaI showed possible mixed Sarcocystis infection. Moreover, the Cryptosporidium genotypes detected were C. parvum (IIdA19G1 and IIaA15G1R1), Cryptosporidium rat genotype IV and a novel genotype (camel genotype). This approach revealed the unique Cryptosporidium genotypes infecting the investigated camels, and the high genetic diversity of the investigated parasites.

Genetic variability within isolates of Sarcocystis species infecting sheep from Egypt.

In order to explain the exact role of sarcocystosis, principally Sarcocystis tenella, in losses of the Egyptian sheep industry, a precise confirmation about the existence of different Sarcocystis species infecting that economically important animal is needed. Therefore, this work aimed to molecularly identify, as well as illustrate the genetic variability within isolates of Sarcocystis spp. infecting sheep from Egypt. Tissue specimens were collected from sheep slaughtered at 3 Egyptian provinces; Cairo, Dakahlia and Damietta. DNA was isolated from the harvested bradyzoites after peptic digestion for the positive sarcocysts infected specimens, and then PCR amplification using the 18S rRNA gene was carried out. PCR products were subjected to gel electrophoresis. DNA from 600 bp gel bands was purified and sequenced. The revealed sequences were compared to their similarities on Genbank, and analyzed both clusterally and phylogenetically. Two Sarcocystis spp. were identified, the macroscopic cyst forming S. gigantea and the microscopic cyst forming S. tenella. Nine S. tenella sequences were analyzed, resulting in 3 polymorphic sites as well as 3 different haplotypes. Clustering of the nine obtained S. tenella sequences in addition to another 23 S. tenella sequences on Genbank revealed low nucleotide (0.001780) diversity as well as negative value of the Taijma neutral index which are indicators for population expansion. Alignment and Phylogeny results illustrated very close relationship between S. gigantea and S. moulei, a goat specific species which rarely reported in sheep, and in turn proposed the cross transmission of the later species between sheep and goats.

Serological diagnosis of Trichinella spiralis in experimentally infected mice.

ELISA and IFA tests were used to detect IgG levels in sera of experimentally infected mice with Trichinella spiralis, during a period of 12 weeks post infection. A crude saline extract of muscle larvae was used for application of ELISA technique, while the intact T. spiralis muscle larvae were used as an antigen for IFAT. In both tests, T. spiralis IgG antibodies could be detected one week post infection in sera of all infected mice. ELISA test showed a sensitivity of 100% during the whole period of the experiment, while its specificity was 93.3%. Meanwhile, IFA test revealed a sensitivity of 100% and a specificity of 85%.