Seroprevalence and molecular detection of Leishmania spp. in cats of West Aegean Region, Turkey.

Zoonotic visceral leishmaniasis is caused by protozoa of the genus Leishmania. Although dogs are considered to be primary reservoir hosts of Leishmania spp., several mammals, such as foxes, jackals and small rodents may also be hosts of different Leishmania spp. Previously, cats were considered as asymptomatic hosts of the parasite without acting as a reservoir. In recent years, there has been an increasing number of leishmaniasis cases in cats, especially in regions where the infection endemically occurs. This data indicate that cats are more likely to be one of the main reservoirs of Leishmania spp. rather than being a non-reservoir host. The aim of this study is to determine the prevalence of Leishmania spp. by molecular and serological techniques among owned and stray cats in four different cities located in western part of Turkey. A total of 386 blood and 301 serum samples were collected from cats in Western Turkey where leishmaniasis is endemic. Feline serum samples were tested by IFAT to detect IgG antibodies against Leishmania spp. antigens. Blood samples collected from 386 cats were examined by PCR for the presence of Leishmania spp. According to PCR results using RV1/RV2 primers, nine (2.3%) out of 386 samples were positive for Leishmania spp. Further PCR analysis using MC1/MC2 primers showed that one cat in Izmir was found to be infected with L. infantum/donovani complex. Sequence analysis revealed the presence of L. infantum, L. major and L. tropica among sampled cats in western part of Turkey. On the other hand, IFAT results indicated that an overall of 47 out of 301 (15.6%) cats that examined by PCR were found to have anti-Leishmania antibodies. Also, six of the seropositive cats were owned animals. The present study demonstrated that both owned and stray cats can be infected with Leishmania spp. and might be potential reservoirs for other animals and humans. Therefore, all communities living in or nearby endemic regions should be made aware of the role of cats as potential reservoirs of infection. In endemic regions, all animals should be protected against infection with insecticides and monitored routinely to control the spread of zoonotic visceral leishmaniasis.

Comparison of protectiveness of recombinant Babesia ovis apical membrane antigen 1 and B. ovis-infected cell line as vaccines against ovine babesiosis.

Babesiosis is a disease complex caused by unicellular Babesia parasites and among them, malignant ovine babesiosis caused by B. ovis has a devastating economical impact on the small ruminant industry. The control of disease is mainly based on chemotherapy and preventing animals from tick infestation and to date no vaccine is available against ovine babesiosis. The requirement for vaccination against B. ovis infection in endemically unstable regions is necessary for implementation of effective disease control measures. The aim of the present study was to evaluate the effectiveness of different immunisation protocols against disease in sheep experimentally vaccinated with recombinant B. ovis apical membrane antigen-1 (rBoAMA-1) and/or live, a B. ovis-infected cell line. Sheep were divided into four experimental groups, plus a control group. Animals were immunised either with the B. ovis stabilate, or with rBoAMA-1, or with both rBoAMA-1 and the B. ovis stabilate. Western blots and ELISAs indicated that immunisation with rBoAMA-1 resulted in generation of a specific response against the recombinant protein, but the degree of antibody response did not correlate with the level of induced protection against challenge. The strongest immune response was induced in animals co-immunised with the live B. ovis stabilate plus rBoAMA-1. Both the hematological and parasitological findings indicated that this co-immunisation regimen has vaccine potential to limit losses incurred by ovine babesiosis in endemic countries.

Infection dynamics of Theileria annulata over a disease season following cell line vaccination.

Tropical theileriosis is a tick-borne haemoparasitic disease of cattle caused by the protozoan parasite Theileria annulata. Globally, the economic impact of the disease is immense and enhanced control measures would improve livestock production in endemic regions. Immunisation with a live attenuated vaccine is an effective and widely used control method, however, the repeated use of live vaccines may have an impact on the field parasite population at a genetic level. Additionally, there has been an increasing number of reports of vaccine breakthrough cases in recent years. Thus, the present study was designed to evaluate the genetic composition of a parasite population over a disease season in a locality where live cell line vaccination is practised. A diverse range of parasite genotypes was identified and every T. annulata positive cattle blood sample harboured multiple parasite genotypes. An alteration in the major genotype and an increasing multiplicity of infection in individual animals was observed over the course of the disease season. Vaccination status was found not to effect within-host multiplicity of infection, while a significantly higher number of genotypes was detected in grazed cattle compared to non-grazed ones. A degree of genetic isolation was evident between parasite populations on a micro-geographic scale, which has not been reported previously for T. annulata. Analysis of parasite genotypes in vaccinated animals suggested only a transient effect of the vaccine genotype on the genetic diversity of the T. annulata population. The vaccine genotype was not detected among clones of two vaccine 'breakthrough' isolates and there is no suggestion that it was responsible for disease. The obtained data indicated that in the system studied there is no apparent risk of introducing the vaccine genotype into the population with only a transient effect on the genetic diversity of the parasite population during the disease season.

Selection of Genetic Markers to Determine Diversity in Theileria annulata Populations after Recombination.

OBJECTIVE: Selecting polymorphic mini- and microsatellite markers to determine genetic diversity and chromosomal regions exhibiting elevated rates of recombination in Theileria annulata populations after recombination. METHODS: The Unipro UGENE software was used to select markers. A score at which 10 times more tandem repeats (TRs) were identified in the real DNA sequence than those in the scrambled sequences of T. annulata was used as the cutoff. TRs containing minimum three nucleotides in length for microsatellite and six nucleotides for minisatellite regions and having a repeat motif identity between 96%-100% with the unit size repeated minimum three times were screened through the whole genome using the suffix array algorithm. RESULTS: A total of 359 minisatellites and 8973 microsatellites were identified. TRs were screened one by one through the whole genome; mini- and microsatellites representing a single region and having suitable regions for primer design were selected based on their localization on T. annulata chromosomes, their repeat motif identity (>96%), and their repeat length (<1500 bp). The primers used to amplify selected candidates were designed, and each primer was used to check 27 different isolates of T. annulata. CONCLUSION: In the present study, a total of 13 polymorphic mini- and microsatellite markers located on the different chromosomes were selected to determine the population diversity of T. annulata.

Determination the Role of Rhipicephalus sanguineus for Transmission of Leishmania major to Reservoir Animals.

OBJECTIVE: This study aimed to explore the role of Rhipicephalus sanguineus in the transmission of Leishmania major, the etiological agent of zoonotic cutaneous leishmaniasis. METHODS: Ten gerbils (Meriones unguiculatus) were infected with promastigotes of L. major, and 10 gerbils were maintained as controls. In a controlled environment, 2000 R. sanguineus larvae were fed to two gerbils. Following feeding to gerbils, 65 tick pools were prepared from the engorged larvae and molted unfed nymphs. These pools were tested for the presence of L. major using polymerase chain reaction and real time (RT) PCR. RESULTS: One of the infected gerbil was anesthetized and necropsied following the dropping of all fed larvae. Following the examination, amastigotes were detected in all organs and tissues. PCR and RT-PCR were performed to test whether the engorged R. sanguineus larvae successfully took the parasite while feeding and was able to transmit it to the next nymphal stage; however, none of the tick pools were found to be positive for L. major. CONCLUSION: Although L.major was not detected in ticks that fed on gerbils, using dogs in experimental studies related to leishmaniasis will give clearer results in terms of detecting the potential role of insects and acars.

[Gerbils, As Experimental Animals (Meriones unguiculatus): Is A Good Role Model for Leishmania major?]. / Deney Hayvani Olarak Gerbiller (Meriones unguiculatus): Leishmania major için Iyi Bir Rol Model Olabilir mi?

OBJECTIVE: This study aimed to observation the possible visceralization tendency and dissemination of L. major amastigotes in gerbils (Meriones unguiculatus) using a classic smear technique, inoculated into enriched Novy-MacNeal-Nicolle (NNN) culture and polymerase chain reaction (PCR) assay for diagnosis of infection. METHODS: In this study, L. major isolated from a man who 18 years old, living in Bitlis province of Turkey. This strain also was utilized to infect gerbils. A total of 1 × 10(8)/mL promastigotes were inoculated to 10 gerbils. Necropsy was performed on infected gerbils for monitoring the visceralization tendency of the parasites. Tissue samples were prepared from each animal and stained by Giemsa and inoculated into NNN culture. However, a real-time PCR assay was performed to confirm the infection the clinical material. RESULTS: Examination of Giemsa-stained tissue smears showed that infected animals with L.major were positive for Leishmania amastigotes in all tissues at the first month post infection and Leishmania promastigotes were cultured at 26°C in culture flasks containing NNN. Melting curve analyses of ribozomal internal transcribed spacer 1 (ITS-1) PCR showed the peak concordant with L. major. CONCLUSION: As a result, the present study confirmed by both Giemsa-stained smears and PCR, visceralization and dissemination of L. major amastigotes, the principal cause of zoonotic cutaneous leishmaniasis in gerbils.