Determination of prevalence and risk factors for infection with <Emphasis Type="Italic">Babesia ovis</Emphasis> in small ruminants from Turkey by polymerase chain reaction

In this study, PCR and thin blood smear-based diagnostic methods were used to assess the frequency of Babesia infection in small ruminants. A total of 300 sheep and 100 goats from 37 randomly selected herds located in eight locations of eastern Turkey were examined for the presence of Babesia infection and any tick species on the body of the animals. Of 400 blood samples examined, 6 (1.5%) were positive for Babesia spp. piroplasms upon microscopic examination, whereas 33 (8.25%) were positive for the presence of B. ovis by PCR. The prevalence of babesiosis in small ruminants detected by PCR was significantly higher than obtained in microscopic examination of thin blood smears (P < 0.05). Thirty-three animals produced the DNA fragment specific for Babesia ovis of which 32 (10.66%) were sheep and 1 (1%) was goat. The difference between the prevalence of Babesia infection in sheep and goats were statistically significant (P < 0.05). The prevalence of Babesia infection in different age groups of sheep were statistically non-significant (P > 0.05). The frequency of B. ovis infection was higher in herds with tick burden than no tick burden (P < 0.05). Seven amplicons (six from sheep and one from goat) were sequenced. The resulting sequences were identical to the recently reported nucleotide sequence of B. ovis. A total of 510 ticks belonging to the Rhipicephalus spp. were collected from sheep. Ticks were identified to be R. bursa and R. turanicus on the basis of morphological features. Nucleotide sequences data reported in this paper are available in EMBL, GenBank and DDJB databases under the accession numbers: DQ409332, DQ409333, DQ409334, DQ409335, DQ409336, DQ409337, DQ409338.     

Biometrical and genetical characterization of large <Emphasis Type="Italic">Babesia Ovis</Emphasis> in Iran

One species of Babesia was identified on the blood smear of 20 different naturally infected sheep in the Northwest of Iran. It was polymorphic, including double pyriform with acute or obtuse angle, single pyriform, and ring form. The size of typical paired pyriforms with acute angle was 2.7 × 0.4 μm (n = 10) and with obtuse angle was 3.5 × 0.6 μm (n = 10). Although the morphological and biometrical parameters resembled the Babesia motasi, the results of seminested polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism using primers specific for small subunit of 18S rRNA confirmed this species as Babesia ovis. Furthermore, the sequence analysis of hypervariable region of small subunit of 18S rRNA revealed the corresponding sequences for B. ovis as well. Experimental infection of healthy lambs with the morphological larger B. ovis showed a milder clinical signs compared to the small one.     

Mitochondrial 16S rDNA sequences and phylogenetic relationships of species of Rhipicephalus and other tick genera among Metastriata (Acari: Ixodidae)

The mitochondrial 16S rRNA gene sequences of the following eight European Metastriata tick species were obtained by direct polymerase-chain-reaction cycle sequencing and silver-staining methods: Rhipicephalus bursa, R. pusillus, R. sanguineus, R. turanicus, Boophilus annulatus, Dermacentor marginatus, Haemaphysalis punctata, and Hyalomma lusitanicum. This mitochondrial gene seems to be a good marker for the establishment of genetic relationships among closely related tick species, but it does not seem to be useful for comparisons of distantly related taxa. The molecular data provide very strong support for the monophyly of the Rhipicephalinae, including Hyalomma spp. However, the genus Rhipicephalus may not be considered a monophyletic group; in all analyses carried out in this study, R. bursa clustered with Boophilus spp. The high percentage of similarity (98.7%) observed between R.␣sanguineus and R. turanicus sequences would suggest that these species recently diverged within the Rhipicephalus genus. Phylogenetic analyses showed a monophyletic relationship among Amblyomminae taxa. The relationships between Haemaphysalis species and the true placement of this genus within Metastriata could not be resolved. Received: 12 September 1997 / Accepted: 3 December 1997     

Detection of bacteria related to <Emphasis Type="Italic">Candidatus</Emphasis> Midichloria mitochondrii in tick cell lines

Many ticks have been shown to be infected with intracellular bacteria. One of these bacteria is Candidatus Midichloria mitochondrii which is the only characterized bacterium that has the ability to invade the mitochondria within ovarian cells and consume them without any effect on the female tick’s reproduction. In the present study, eight cell lines derived from the ticks Ixodes ricinus, Ixodes scapularis, Rhipicephalus (Boophilus) microplus, and Rhipicephalus (Boophilus) decoloratus were examined for the presence of the bacterium Ca. Midichloria mitochondrii. PCR assays for this bacterium were carried out using two sets of primers targeting the eubacterial 16SrRNA gene and a set of primers specific for the gyrB gene of Ca. Midichloria mitochondrii. With the 16S rRNA primers, DNA was amplified from two cell lines (R. (B.) decoloratus line BDE/CTVM14 and I. ricinus line IRE/CTVM19) on one out of three occasions each. Sequencing of the PCR products showed that the two cell lines gave sequences with 100% similarity to Ca. Midichloria mitochondrii. However, all cell lines, including the two positive cell lines, were negative with the specific primers. Phylogenetic analysis shows that our sequences belong to the subclass α-proteobacteria. They were identical to the sequences amplified from the tick I. ricinus. The results suggest that two cell lines, IRE/CTVM19 and BDE/CTVM14, may contain bacteria closely related to Ca. Midichloria mitochondrii and identical with it in a 350-bp part of the 16S rRNA gene sequence. To our knowledge, this constitutes the first report of the presence of DNA similar to the DNA of Ca. Midichloria mitochondrii in tick cell lines.     

Simultaneous detection and differentiation of<Emphasis Type="Italic"> Theileria</Emphasis> and<Emphasis Type="Italic"> Babesia</Emphasis> parasites infecting small ruminants by reverse line blotting

Characteristic sequence signatures were identified within the hypervariable region 4 (V4 region) of the small ribosomal RNA gene of ovine/caprine piroplasm species including Theileria lestoquardi, T. ovis , T. separata , Babesia ovis , B. motasi , B. crassa [comprising strains B. crassa (Iran) and B. crassa (Turkey)] and several novel species: Theileria sp. 1 (China), Theileria sp. 2 (China) and Babesia sp. (China), [comprising strain Babesia sp. (Lintan), and Babesia sp. (Ningxian)] as defined previously. Based on the ascertained gene variations a reverse line blotting (RLB) assay was developed enabling direct, concurrent, highly specific and sensitive identification of virtually all presently known ovine/caprine piroplasm species. All probes bound to their respective target sequence only, therefore, no cross-reaction was observed resulting in clear recognition of either individual strains, species or groups. No signal was observed when ovine and caprine genomic DNA was used as the control, demonstrating that the signals are due to the presence of parasite DNA in investigated samples. Furthermore, the sensitivity of RLB could be considerably enhanced to detect a parasitemia level of at least 10^-12% by reamplification of PCR products (nested PCR) thereby substantially increasing the possibility of identifying carrier animals.     

Canine babesiosis in Romania due to <Emphasis Type="Italic">Babesia canis</Emphasis> and <Emphasis Type="Italic">Babesia vogeli</Emphasis>: a molecular approach

Canine babesiosis is a tick-borne disease caused by the protozoa Babesia spp. that affects dogs worldwide. In Romania, canine babesiosis has become quite frequent in the last few years, with a wide variety of clinical signs, ranging from mild, nonspecific illness to peracute collapse, and even death. Traditionally, a Babesia infection in dogs is diagnosed based on the morphologic appearance of the intraerythrocytic piroplasms observed in peripheral blood smears. To date, no data on genetic characterization of Babesia species in dogs has been documented for Romania. Therefore, a molecular survey on natural Babesia infections of dogs in Romania using polymerase chain reaction and genetic sequence analysis of a fragment of the ssRNA gene was performed. A total number of 16 blood samples were tested for the presence of Babesia DNA. Blood samples were collected from 11 dogs with symptoms of babesiosis and microscopically proven positive for Babesia and from a group of five asymptomatic dogs, not tested microscopically for Babesia, which were included in the study for comparative analysis. The piroplasm-specific PCR amplifying the partial 18S rRNA gene confirmed Babesia spp. infection in all 11 samples from dogs with clinical babesiosis, and in one of the clinically normal dogs. Sequence analysis revealed the presence of Babesia canis in all clinically affected dogs and Babesia vogeli in one clinically normal dog. This is the first molecular evidence of B. canis and B. vogeli in dogs from Romania. The results of the study provide basic information toward a better understanding of the epidemiology of canine babesiosis in Romania and will help to promote an effective control program.     

Spotted fever group <Emphasis Type="Italic">Rickettsia</Emphasis> in brown dog ticks <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> in southwestern Spain

A total of 2,229 adults ticks (1,428 males and 801 females) belonging to the brown dog tick, Rhipicephalus sanguineus Latreille, 1806, collected from dogs in Seville province (Andalusia), distributed in 500 lots ranging from one to eight specimens per lot, were examined for the presence of rickettsiae by molecular techniques. Specific rickettsiae DNA were detected in 90 lots (18%) of ticks tested. Sequence analysis of amplicons revealed that R. sanguineus ticks were infected exclusively with Rickettsia massiliae (including the strain Bar-29). The results of this study extend the knowledge of the geographic distribution and prevalence of these spotted fever group (SFG) rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in brown dog ticks in south western Spain. Although Mediterranean spotted fever (MSF) is an endemic disease in Andalusia, Rickettsia conorii was not found, whereas R. massiliae, recently described as a pathogenic species, was highly prevalent in this area. Our data suggest that in Andalusia a number of MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsia present in R. sanguineus, particularly, R. massiliae.     

An evaluation of primers amplifying DNA targets for the detection of <Emphasis Type="Italic">Cryptosporidium</Emphasis> spp. using <Emphasis Type="Italic">C. parvum</Emphasis> HNJ-1 Japanese isolate in water samples

The performance of polymerase chain reaction (PCR) procedures for the detection of Cryptosporidium parvum HNJ-1 strain (genotype II) oocysts purified from mice using published protocols was evaluated. Oocysts were concentrated from fecal samples of infected severe combined immunodeficiency (SCID) mice by sucrose flotation and were then purified by immunomagnetic separation method. The genotype of C. parvum was established as type II by restriction fragment length polymorphism (RFLP) analysis. Water samples were spiked with different numbers of oocysts, determined by limiting dilution. Genomic DNA was extracted and used for PCR assays targeting various Cryptosporidium species genes (Beta-Tubulin, COWP, 70 kDa HSP, SSU rRNA, ITS1, TRAP-C1 and TRAP-C2 gene). DNA from oocyst numbers of more than 1 × 10⁴ was detected using each of the primers. However, when using lower oocyst numbers, the tools based on 9 of the 16 different primer assays gave sufficient results. Assays using the remaining seven primers gave less than satisfactory results. A new primer set, named VKSS-F1/2 and VKSS-R1/2, that target the 18 SSU rRNA gene of C. parvum was constructed and applied.The VKSS-F1/2 and VKSS-R1/2 assays amplified DNA isolated from spiked samples in 206 of 211 trials (97.6%). This illustrates the difficulty of detecting low numbers of Cryptosporidium spp. oocysts by molecular methods when working with environmental samples.     

Deletion of the <Emphasis Type="Italic">MAT-</Emphasis>2 mating-type gene during uni-directional mating-type switching in <Emphasis Type="Italic">Ceratocystis</Emphasis>

 Ceratocystis eucalypti is strictly heterothallic, with single ascospore strains representing one of two opposite mating types. Most other Ceratocystis species, including C. virescens, C. pinicola, and C. fimbriata, are homothallic. In the homothallic species, the MAT-2 strains are self-fertile, while MAT-1 strains are self-sterile and grow more slowly than MAT-2 strains. The current hypothesis is that self-fertility of MAT-2 strains is due to the deletion of the MAT-2 mating-type gene, resulting in the expression of the MAT-1 mating type. These mutant MAT-1 strains are able to cross with MAT-2 strains. Part of the MAT-2 mating-type gene in C. eucalypti, C. pinicola, and C. fimbriata was amplified using degenerate primers designed from the conserved MAT-2 HMG DNA-binding motif. The expected approximately 300-bp PCR products were cloned and sequenced. Specific primers were designed that amplified 210-bp fragments only in MAT-2 isolates of C. eucalypti, C. virescens, C. pinicola, and C. fimbriata. These fragments were present in self-fertile field isolates and self-fertile progeny but were absent in the self-sterile (MAT-1) progeny from selfings of C. virescens, C. pinicola, and C. fimbriata, thus supporting the hypothesis that the MAT-2 mating-type gene is deleted during uni-directional mating-type switching. A Southern-blot analysis was performed to confirm the deletion of MAT-2 gene in self-sterile progeny. The DNA sequence data for the C. eucalypti MAT-2 mating-type gene was increased to 1371-bp using TAIL-PCR and uneven PCR, representing a portion of the complete MAT-2 gene DNA sequence. Received: 5 November 1999 / 25 February 2000     

Discrimination of <Emphasis Type="Italic">Cryptosporidium</Emphasis> species by denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) was used for the discrimination of three species and one genotype of the protozoan parasite Cryptosporidium: the C. parvum, C. andersoni, C. muris, and C. muris Japanese field mouse genotype. A set of primers specific for the 18S rRNA gene of Cryptosporidium was used in the DGGE; consequently, the four strains showed different banding patterns. This is a potentially convenient and precise method for the discrimination of Cryptosporidium spp.     

Molecular detection and identification of Ehrlichia and Anaplasma species in ixodid ticks

A polymerase chain reaction (PCR) assay followed by partial sequencing of the 16S ribosomal RNA gene was performed for the presence of Ehrlichia and/or Anaplasma. A total of 242 ixodid ticks were collected from domestic ruminants and their shelters, as well as humans, and their individual salivary glands were dissected out for DNA. From the 242 ticks analyzed, six (2.47%), comprising three Hyalomma anatolicum anatolicum, one Rhipicephalus bursa, and two Rhipicephalus sanguineus, were positive. Of these sequenced samples directly obtained from the PCR products, three sequences from H. a. anatolicum were identical to that of the gene of Ehrlichia spp. strains. One sequence identified in R. bursa was closely related to Anaplasma platys. The remaining two sequences detected in R. sanguineus were similar to that of the gene of Anaplasma ovis. The study presented here provides preliminary data regarding the presence of rickettsial pathogens in ticks in Turkey. Nucleotide sequence data reported in this paper are available in GenBank, EMBL, and DDBJ databases under accession numbers EU191227–EU191231 and EU191233.     

Phylogenetic analysis of<Emphasis Type="Italic"> Theileria</Emphasis> species transmitted by<Emphasis Type="Italic"> Haemaphysalis qinghaiensis</Emphasis>

The phylogenetic relationships between six isolates of Theileria spp. infective to small ruminants, and two isolates of Theileria spp. infective to yak, all transmitted by Haemaphysalis qinghaiensis, together with the Theileria orientalis/sergenti/buffeli group and T. sinensis, were analyzed using the 18S ssrRNA gene sequence. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was used either for direct sequencing or was ligated to the PCR II vector for sequencing. The length of the 18S ssrRNA gene of all Theileria spp. involved in this study was around 1,740 bp. Two phylogenetic trees were inferred based on the 18S ssrRNA gene sequence of the Chinese isolates only, and Chinese isolates and other species of Theileria available in GenBank. In the first tree, the Theileria sp. infective to yaks was found to be T. sinensis. The Theileria sp. infective to small ruminants was found to be composed of two separate species of Theileria. Theileria sp. from Qinghai, Madang, Ningxian and Lintan, which was identical to the unidentified Theileria sp. described previously, is designated Theileria sp (China 1). The Theileria sp. from Longde, Zhangjiachuan and Lintan, which has not been described previously, is designated Theileria sp. (China 2) in order to avoid confusion. In the second tree, Theileria sp. (China 1) was closely related to benign Theileria, such as T. buffeli and T. sergenti, while Theileria sp. (China 2) was separated from other Theileria spp. The results indicate that H. qinghaiensis transmit at least three species of Theileria, two which are infective to sheep and goats, but not yak and one which is infective to yaks and cattle, but not to sheep and goats.     

Genotypically unique <Emphasis Type="Italic">Babesia </Emphasis>spp. isolated from reindeer (<Emphasis Type="Italic">Rangifer tarandus tarandus</Emphasis>) in the United States

Two morphologically dissimilar Babesia spp. were cultured from reindeer (Rangifer tarandus tarandus) in Placer County, Calif. The smaller isolate, designated RD61, was morphologically similar to Babesia odocoilei. Serum from RD61-infected reindeer reacted equally strongly to B. odocoilei and RD61 parasites in the indirect fluorescent antibody (IFA) test. Small subunit ribosomal RNA (SSU rRNA) gene-sequence analysis showed 99.0% identity to that of B. odocoilei. The larger piroplasm, designated RD63, resembled larger babesia organisms, such as Babesia caballi and Babesia bigemina. Serum from RD63-infected reindeer also reacted with both B. odocoilei and RD61 parasites in the indirect fluorescent antibody test. The SSU rRNA gene showed 94.2% identity to that of B. bigemina. Further studies are needed to determine whether these parasites are the same as the Babesia spp. previously documented in Siberian reindeer.     

Identification of<Emphasis Type="Italic"> Borrelia burgdorferi</Emphasis> sensu lato,<Emphasis Type="Italic"> Anaplasma</Emphasis> and<Emphasis Type="Italic"> Ehrlichia</Emphasis> Species,and Spotted Fever Group Rickettsiae in Ticks from Southeastern Europe

Prevalence data for tick-borne pathogens are used to assess the risk for human health. In this study the presence and identity of Borrelia burgdorferi sensu lato, Ehrlichia, Anaplasma, and Rickettsia species in Bulgarian Ixodes ricinus ticks and in non-Ixodes ticks from Turkey and Albania was determined by polymerase chain reaction (PCR) and reverse line blot hybridization. In the adult Bulgarian ticks, the prevalence of Borrelia burgdorferi sensu lato infection was approximately 40%, while Borrelia afzelii was the predominant species, representing more than half of all Borrelia-positive ticks. Ehrlichia and Anaplasma species were detected in 35% of the adult Ixodes ricinus ticks and in 10% of the nymphs. Sequence analysis of PCR products reacting with the Anaplasma phagocytophila probe revealed a 16S rRNA gene identical to that of the Anaplasma phagocytophila prototype strain. Ehrlichia and Anaplasma species were found in approximately 7% of the non-Ixodes ticks. Sequence analysis of some of these samples revealed the presence of Anaplasma ovis, Ehrlichia canis, and a species closely resembling Ehrlichia chaffeensis. About half of all adult ticks examined and approximately 20% of all nymphs were infected with Rickettsia species. In Ixodes ricinus ticks, Rickettsia helvetica and a Rickettsia species designated as IRS3 were found in high prevalence. Rickettsia conorii was found in virtually all non-Ixodes tick species from Albania and Turkey. The results of this study show that many tick-borne diseases are most probably endemic in the Balkan area. Furthermore, the results suggest that there is a considerable chance for simultaneous transmission of tick-borne pathogens to human beings.     

Detection and molecular characterization of <Emphasis Type="Italic">Babesia caballi</Emphasis> and <Emphasis Type="Italic">Theileria equi</Emphasis> isolates from endemic areas of Brazil

Blood samples were collected from 487 adult horses, including 83 pregnant mares, at a slaughterhouse located in Araguari, Minas Gerais State, Brazil. For each blood sample, the packed cell volume (PCV) was determined, and Giemsa-stained smears were microscopically examined for the presence of hemoparasites. The plasma was examined by the indirect fluorescent antibody test for detection of antibodies against Babesia caballi and Theileria equi. In addition, DNA was extracted and analyzed by a multiplex real-time polymerase chain reaction (PCR), specific for B. caballi and T. equi. Products of PCR were sequenced and compared with each other and with known sequences. The serological results showed a total prevalence of 91.0% for T. equi and 83.0% for B. caballi, while by PCR, prevalences of 59.7% for T. equi and 12.5% for B. caballi were observed. However, no correlations were seen between positivity (neither by serology nor by PCR) and PCV values. As expected, the microscopic examination of blood smears showed low sensitivity in detecting the infections when compared to the PCR. Only 35 out of 570 blood smears were positive, with parasitemias below 0.1%. No congenital transmission was detectable. As far as sequencing is concerned, no differences were seen among the isolates of each species nor among them and known sequences available. These results confirm, by molecular methods, the high prevalence rates of T. equi and B. caballi infections in carrier horses in Brazil. However, no diversity was observed among the isolates within the studied regions.     

Molecular identification of <Emphasis Type="Italic">Trichuris vulpis</Emphasis> and <Emphasis Type="Italic">Trichuris suis</Emphasis> isolated from different hosts

Trichuris suis was isolated from the cecum of two different hosts (Sus scrofa domestica—swine and Sus scrofa scrofa—wild boar) and Trichuris vulpis from dogs in Sevilla, Spain. Genomic DNA was isolated and internal transcribed spacers (ITS)1-5.8S-ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced using polymerase chain reaction techniques. The sequence of T. suis from both hosts was 1,396 bp in length while that of T. vulpis was 1,044 bp. ITS1 of both populations isolated of T. suis was 661 nucleotides in length, while the ITS2 was 534 nucleotides in length. Furthermore, the ITS1 of T. vulpis was 410 nucleotides in length, while the ITS2 was 433 nucleotides in length. One hundred fifty-four nucleotides were observed along the 5.8S gene of T. suis and T. vulpis. Intraindividual and intraspecific variations were detected in the rDNA of both species. The presence of microsatellites was observed in all the individuals assayed. Sequence analysis of the ITSs and the 5.8S gene has demonstrated no sequence differences between T. suis isolated from both hosts (S. scrofa domestica—swine and S. scrofa scrofa—wild boar). Nevertheless, clear differences were detected between the ITS1 and ITS2 of T. suis and T. vulpis. Furthermore, a comparative molecular analysis between both species and the previously published ITS1-5.8S-ITS2 sequence data of Trichuris ovis, Trichuris leporis, Trichuris muris, Trichuris arvicolae, and Trichuris skrjabini was carried out. A common homology zone was detected in the ITS1 sequence of all species of trichurids.     

Phylogeny of sheep and goat<Emphasis Type="Italic"> Theileria</Emphasis> and<Emphasis Type="Italic"> Babesia</Emphasis> parasites

The phylogenetic relationship of Theileria and Babesia species infecting sheep and goats on the basis of their 18S RNA gene structure was addressed in the present study. For this purpose, the complete sequences of the small ribosomal RNA genes of a panel of sheep and goat piroplasm isolates, including T. lestoquardi, T. ovis, T. separata, B. ovis, B. motasi, B. crassa and several novel species, were sequenced and compared. The classification based on the established phylogenetic tree corresponded with traditional systematics and revealed that sheep/goat piroplasm species are of polyphyletic origin. The independent evolution of almost all sheep/goat piroplasms suggests that speciation may have occurred after transfer of the piroplasm-transmitting tick from a primal wild ruminant host to domestic sheep and goats. In accordance with recent reports, our study confirms the existence of at least two additional sheep/goat piroplasm species, designated Theileria sp. 1 (China) and Theileria sp. 2 (China). The recently reported pathogenic sheep/goat Theileria sp. 1 (China) seems to be identical with a Theileria sp. isolated from Japanese serow. Furthermore, our results suggest that T. ovis represents a single species.     

<Emphasis Type="Italic">Hepatozoon canis</Emphasis> infection in ticks during spring and summer in Italy

Hepatozoon canis is a common protozoan of dogs, being among the most prevalent tick-borne pathogens infecting dogs around the world. It is primarily transmitted by Rhipicephalus sanguineus, the brown dog tick. In this study we tested ticks collected from dogs and from the environment in order to track the origin of an outbreak of H. canis infection detected in October 2009 in a private dog shelter in southern Italy. Ticks from dogs (n = 267) were collected during the spring of 2009, whereas ticks from environment (n = 300) were found on sticky traps placed in the same shelter during the summer of 2009. All ticks were tested by PCR for the detection of a H. canis 18S ribosomal RNA gene fragment. Four (1.5%, one female and three males) ticks collected from dogs were PCR positive. None of the larvae collected from the environment were positive, but a relatively high infection rate (8.0%) was detected in nymphs. These findings point out that dogs became infected during the summer, when ticks were abundant and highly infected by H. canis. Moreover, this study suggests that castor oil sticky traps might be useful to collect engorged immature ticks in highly infested environments (e.g., dog shelters). This might be particularly interesting to evaluate the level of infection by certain pathogens in free-ranging ticks R. sanguineus, as done in the present study.     

<Emphasis Type="Italic">Sarcocystis</Emphasis> sp. from the herring gull (<Emphasis Type="Italic">Larus argentatus</Emphasis>) identity to <Emphasis Type="Italic">Sarcocystis wobeseri</Emphasis> based on cyst morphology and DNA results

Having studied 11 herring gulls (Larus argentatus) Sarcocystis cysts were found in neck and leg muscles of 4 birds. One type of sarcocysts (cyst type I) that have a thin (∼1.0 μm), smooth, or slightly wavy cyst wall without clearly visible protrusions and small (6.0–8.0 μm) lancet- or banana-shaped cystozoites was identified by the light microscopy. Sarcocysts extracted from one herring gull were used for electron microscopy and DNA analysis. Ultrastructurally, Sarcocystis sp. from the herring gull had the same tissue cyst wall type-1 as S. calchasi, S. columbae, and S. wobeseri parasitizing in birds. According to first internal transcribed spacer (ITS-1) region, 18S rRNA and 28S rRNA gene sequences, Sarcocystis sp. from the herring gull belongs to S. wobeseri. Nevertheless, without evidences of cross-transmission experiment sarcocysts extracted from herring gull at present time are named as S. wobeseri-like.     

Molecular evidence of <Emphasis Type="Italic">Chlamydiales</Emphasis> in ticks from wild and domestic hosts in Sardinia,Italy

Ticks are well known to be important vectors for a wide range of bacteria, viruses and protozoa affecting human and animal health. Ixodid ticks are widely distributed in Sardinia, and an increasing number of tick-borne bacteria have been documented in the island. A growing number of evidence are supporting the hypothesis of alternative transmission routes for chlamydial bacteria such as the involvement of vectors. This study was conducted to provide possible molecular detection of members belonging to the Chlamydiales order in Sardinian ticks and to update information concerning the presence of new ectoparasite-borne bacteria in ticks collected from domestic and wild hosts in a typical Mediterranean environment. A total of 378 ticks were individually screened with a pan-Chlamydiales specific primers targeting the 16S rRNA gene. Chlamydiales DNA was detected in 28% of the total ticks analyzed. The analyses of sequences highlighted that Rhipicephalus sanguineus sensu lato, Rhipicephalus bursa, Rhipicephalus annulatus, Haemaphysalis sulcata, Haemaphysalis punctata and Dermacentor marginatus ticks exhibited DNA of Chlamydiaceae and Parachlamydiaceae members. Our results revealed that DNA of zoonotic microorganisms such as C. psittaci, C. abortus and the emerging pathogen Parachlamydia acanthamoebae are present in Sardinian ticks. Since routes of Chlamydia transmission are yet to be fully defined, the role of ticks as possible vectors for Chlamydiales remains the most challenging and interesting question to be addressed in future research. Continued monitoring of these pathogens in tick vectors is needed to provide strategies for controlling of possible chlamydial infections and disease outbreaks in the island.