Yaarit Nachum-Biala

B‐vitamins for neuroprotection: Narrowing the evidence gap

A compelling and extensive epidemiological literature documents the strong association of inadequate status of folate, vitamin B12, and to a lesser degree vitamin B6, with increased risk of neurodegenerative and cerebrovascular disease. Mildly elevated plasma total homocysteine, which is biochemically related to low status of these B‐vitamins, is similarly associated with increased risk for these conditions. This, together with experimental data showing that experimental B‐vitamin deficiency and/or hyperhomocysteinemia can cause a variety of neurological and vascular deficits in animals, has provided the evidence base and motivation for a growing number of large randomized, double‐blind clinical trials aimed at determining the efficacy and safety of B‐vitamin supplementation for preserving cognitive function in older adults. Despite some encouraging trials showing benefit of B‐vitamins for slowing brain atrophy and cognitive decline, the majority of these studies have not demonstrated that B‐vitamin supplementation has protective or therapeutic cognitive benefit. There are many possible explanations for the inconsistency between the clinical trials and for the discrepancy between their findings and the predictions of the epidemiological evidence. Among these are the possibility of inadequate hypotheses guiding the trials, design limitations of the individual trials, and inherent limitations of nutritional randomized clinical trials. Resolving these issues will be crucial for designing definitive trials and ultimately for guiding nutritional interventions for cognitive protection.

First report on Babesia cf. microti infection of red foxes (Vulpes vulpes) from Hungary

BackgroundTo date, only one report of a small Babesia infection based on microscopic observation which caused babesiosis in two dogs in Hungary has been published. Babesiosis due to Babesia canis - which is endemic in the local dogs - has only been detected in captive grey wolves. No information is available on babesial/theilerial infections in red foxes in Hungary. The aim of the study was to screen red foxes in Hungary for babesial parasites by PCR and to compare their partial 18S rRNA gene sequences to those parasites of domestic dogs and wild canids from other countries.MethodsBlood samples of 404 red foxes originating from 316 locations representing all 19 Hungarian counties were screened in Hungary for babesial parasites by PCR and the partial 18S rRNA gene sequences were compared to those parasites of domestic dogs and wild canids from other countries.ResultsAltogether 81 red foxes out of 404 (20.0%; 95% CI: 16.4–24.2%) shot in 74 locations and in 17 of the 19 Hungarian counties were found to be infected with Babesia cf. microti by PCR.ConclusionsThis is the first report to demonstrate the occurrence of Babesia cf. microti in Hungary, and its widespread presence in the fox population throughout the country. Further studies are needed to identify the tick species involved in its transmission, and whether other mechanisms of transmission are involved in its spread in fox populations.

Mucocutaneous Leishmania tropica infection in a dog from a human cutaneous leishmaniasis focus

BackgroundLeishmania tropica is a causative agent of cutaneous leishmanaisis in the Middle East, North Africa and parts of southeastern Europe. Although transmission of L. tropica has been reported as anthroponotic, in Israel it was found to have a zoonotic pattern.FindingsA one year old male Pekingese dog from Maale Adumim, a focus of L. tropica human cutaneous leishmaniasis near Jerusalem, was presented by its owner with a large proliferative red mucocutaneous lesion on the lip between the mouth and nose. Physical examination and a biochemistry panel were normal and a complete blood count showed mild leukocytosis with lymphocytosis and eosinophilia. A biopsy of the lesion was suggestive of the presence of Leishmania organisms. Serology for Leishmania sp. by ELISA was positive and an aspirate from the lesion showed a large number of Leishmania amastigotes. ITS1-HRM-PCR of the lesion was positive and sequencing indicated that infection was caused by L. tropica, which was also cultured from the lesion. Blood PCR was negative. The dog responded well to allopurinol treatment and its lesion shrunk considerably within one month of therapy and healed after two months.ConclusionsOnly a few cases of dog infection with L. tropica have been described to date. They were reported from Morocco and Iran and involved infection of visceral organs. This is the first report of focal mucocutaneous L. tropica infection in a dog and its response to anti-leishmanial treatment. Domestic and wild canines should be evaluated for being possible animal reservoirs for human L. tropica infection in endemic areas or merely accidental hosts.

Prevalence study and risk factor analysis of selected bacterial, protozoal and viral, including vector-borne, pathogens in cats from Cyprus

BackgroundFeline infectious agent studies are lacking in Cyprus. The aims of this study were to determine the prevalence and risk factors for various feline infectious agents, including feline vector-borne pathogens (FVBP), in cats from Cyprus.MethodsA cross-sectional, descriptive, multicentre study was performed on 174 feline samples [138 owned and 36 shelter-feral, including both healthy (43) and non-healthy (131), cats] from private veterinary clinics from all six districts of Cyprus. Real-time quantitative polymerase chain reaction (qPCR) assays were used to detect Mycoplasma haemofelis (Mhf), “Candidatus Mycoplasma haemominutum” (CMhm) and “Candidatus Mycoplasma turicensis” (CMt). The population was tested for four FVBP including Bartonella henselae and Leishmania spp. using qPCR, while conventional PCR assays were used to detect Ehrlichia/Anaplasma spp. and Hepatozoon spp. Serological assays were performed to detect Leishmania infantum antibodies, feline leukaemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies. Statistical analysis was performed to test associations and possible risk factors between variables and infectious agents.ResultsNinety-six (55.2%) of the 174 cats were PCR-positive for at least one infectious agent. Forty-six cats (26.4%) were haemoplasma positive, including 13 (7.5%) for Mhf, 36 (20.7%) for CMhm and 12 (6.9%) for CMt. Sixty-six cats (37.9%) were positive for Hepatozoon spp., while 19 (10.9%) were positive for B. henselae, four (2.3%) for Leishmania spp. and one (0.6%) for Ehrlichia/Anaplasma spp. Sequencing revealed the presence of Hepatozoon felis, L. infantum and Anaplasma platys. Of the 164 cats that underwent retroviral serology, 10 (6.1%) were FeLV-positive and 31 (18.9%) were FIV-positive, while L. infantum serology was positive in 7 (4.4%) of the 160 cats tested. Multivariable logistic regression revealed significant associations for various infectious agents including L. infantum with each of Hepatozoon spp. and CMt infection.ConclusionsA high prevalence of infectious agents was found in cats from Cyprus with Mhf, CMhm, CMt, L. infantum, B. henselae, H. felis, A. platys, FeLV and FIV infections reported for the first time. The significant associations between different pathogens provide a better understanding of similarities in the epidemiology of these pathogens and interactions between them.

Molecular detection of emerging tick-borne pathogens in Vojvodina, Serbia

Ticks play an important role in disease transmission globally due to their capability to serve as vectors for human and animal pathogens. The Republic of Serbia is an endemic area for a large number of tick-borne diseases. However, current knowledge on these diseases in Serbia is limited. The aim of this study was to investigate the presence of new emerging tick-borne pathogens in ticks collected from dogs and the vegetation from different parts of Vojvodina, Serbia. A total of 187 ticks, including 124 Rhipicephalus sanguineus, 45 Ixodes ricinus and 18 Dermacentor reticulatus were collected from dogs. In addition, 26 questing I. ricinus ticks were collected from the vegetation, using the flagging method, from 4 different localities in Vojvodina, Serbia. DNA was extracted from each tick individually and samples were tested by either conventional or real-time PCR assays for the presence of Rickettsia spp.-DNA (gltA and ompA gene fragments), Ehrlichia/Anaplasma spp.-DNA (16S rRNA gene fragment) and Hepatozoon spp./Babesia spp.-DNA (18S rRNA gene fragment). In addition, all I. ricinus DNA samples were tested for Bartonella spp.-DNA (ITS locus) by real-time PCR. In this study, the presence of novel emerging tick-borne pathogens including Rickettsia raoultii, Rickettsia massiliae, Babesia venatorum, Babesia microti, Hepatozoon canis and Candidatus Neoehrlichia mikurensis was identified for the first time in Serbia. Our findings also confirmed the presence of Rickettsia monacensis, Babesia canis and Anaplasma phagocytophilum in ticks from Serbia. The findings of the current study highlight the great diversity of tick-borne pathogens of human and animal importance in Serbia. Physicians, public health workers and veterinarians should increase alertness to the presence of these tick-borne pathogens in this country.

Molecular characterization of Theileria orientalis from cattle in Ethiopia.

This study reports the first molecular characterization of Theileria orientalis in local breeds of cattle in Ethiopia. A conventional PCR utilizing major piroplasm surface protein (MPSP) gene and an established multiplexed tandem PCR (MT-PCR) were used to characterize T. orientalis and to assess the infection intensity, respectively. Of 232 blood samples tested, T. orientalis DNA was detected in only 2.2% of samples using conventional PCR; two genotypes buffeli (1.3%; 3/232) and type 5 (0.9%; 2/232) of T. orientalis were detected. Phylogenetic analysis revealed that the buffeli MPSP sequences from Ethiopia were closely related to those reported from Kenya, Sri Lanka and Myanmar, and type 5 sequences from Ethiopia grouped with those from Korea, Japan, Vietnam and Thailand. A higher number of samples (3.9%; 9/232) were test-positive by MT-PCR and four genotypes (buffeli, chitose, ikeda and type 5) of T. orientalis were detected. The average intensity of infections with genotypes buffeli (DNA copy numbers 11,056) and type 5 (7508) were significantly higher (P<0.0001) than the pathogenic genotype ikeda (61 DNA copies). This first insight into T. orientalis from cattle in Ethiopia using MPSP gene provides a basis for future studies of T. orientalis in various agroclimatic zones and of the impact of oriental theilerosis on cattle in this and other countries of Africa.

Relationship between the Presence of Bartonella Species and Bacterial Loads in Cats and Cat Fleas (Ctenocephalides felis) under Natural Conditions.

ABSTRACT Cats are considered the main reservoir of three zoonotic Bartonella species: Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae. Cat fleas (Ctenocephalides felis) have been experimentally demonstrated to be a competent vector of B. henselae and have been proposed as the potential vector of the two other Bartonella species. Previous studies have reported a lack of association between the Bartonella species infection status (infected or uninfected) and/or bacteremia levels of cats and the infection status of the fleas they host. Nevertheless, to date, no study has compared the quantitative distributions of these bacteria in both cats and their fleas under natural conditions. Thus, the present study explored these relationships by identifying and quantifying the different Bartonella species in both cats and their fleas. Therefore, EDTA-blood samples and fleas collected from stray cats were screened for Bartonella bacteria. Bacterial loads were quantified by high-resolution melt real-time quantitative PCR assays. The results indicated a moderate correlation between the Bartonella bacterial loads in the cats and their fleas when both were infected with the same Bartonella species. Moreover, a positive effect of the host infection status on the Bartonella bacterial loads of the fleas was observed. Conversely, the cat bacterial loads were not affected by the infection status of their fleas. Our results suggest that the Bartonella bacterial loads of fleas are positively affected by the presence of the bacteria in their feline host, probably by multiple acquisitions/accumulation and/or multiplication events.

Three different Hepatozoon species in domestic cats from southern Italy

Three species of Hepatozoon, namely, Hepatozoon felis, Hepatozoon canis and Hepatozoon silvestris may affect domestic and/or wild felids. Although hepatozoonosis has been documented in a wide range of mammal species, data on cats are limited. To investigate the occurrence of these pathogens in cats, blood samples were collected from animals living in three provinces of southern Italy (Bari, Lecce, and Matera), and molecularly analysed by PCR amplification and sequencing of segments of the 18S rRNA gene. Out of 196 blood samples collected, Hepatozoon spp. DNA was amplified in ten cats (5.1%, CI: 3%-9%), with the majority of infected animals from Matera (8/34, 23.5%) and one each from the other two provinces. BLAST analysis revealed the highest nucleotide identity with sequences of H. canis, H. felis and H. silvestris deposited in GenBank. Results of this study indicate that these three species of Hepatozoon infect domestic cats in Italy. This is the first report of H. silvestris infection in a domestic cat.

Novel evidence suggests that a 'Rickettsia felis-like' organism is an endosymbiont of the desert flea, Xenopsylla ramesis

Fleas are acknowledged vectors and reservoirs of various bacteria that present a wide range of pathogenicity. In this study, fleas collected from wild rodents from the Negev desert in southern Israel were tested for RickettsiaDNA by targeting the 16S rRNA (rrs) gene. Thirty‐eight Xenopsylla ramesis, 91 Synosternus cleopatrae and 15 Leptopsylla flea pools (a total of 568 fleas) were screened. RickettsiaDNA was detected in 100% of the X. ramesis and in one S. cleopatrae flea pools. None of L. algira flea pools was found positive. All positive flea pools were further characterized by sequencing of five additional genetic loci (gltA, ompB, ompA, htrA and fusA). The molecular identification of the positive samples showed all sequences to be closely related to the ‘Rickettsia felis‐like’ organisms (99–100% similarities in the six loci). To further investigate the association between ‘R. felis‐like’ and X. ramesis fleas, ten additional single X. ramesis adult fleas collected from the wild and five laboratory‐maintained X. ramesis imago, five larva pools (2–18 larvae per pool) and two egg pools (18 eggs per pool) were tested for the presence of ‘R. felis‐like’ DNA. All samples were found positive by a specific ompAPCR assay, confirming the close association of this Rickettsia species with X. ramesis in all its life stages. These results suggest a symbiotic association between ‘Rickettsia felis‐like’ and X. ramesis fleas.

Evaluation of a commercial in-clinic point-of-care polymerase chain reaction test for Ehrlichia canis DNA in artificially infected dogs

A novel in-clinic point-of-care (ICPOC) polymerase chain reaction (PCR) test was evaluated for its ability to detect Ehrlichia canis DNA in artificially infected dogs compared to a real-time PCR assay. Six Beagle dogs negative for E. canis antibodies and PCR negative were artificially infected with an Israeli E. canis strain (611). All dogs developed IgG antibodies 8 days post infection (PI), and clinical and hematological abnormalities on day 10 PI. Only the real-time PCR detected E. canis DNA in the blood of five dogs at days 3 and 5 PI. At day 12 PI during the acute phase of the disease, 1 day after the initiation of doxycycline treatment, the ICPOC PCR assay detected E. canis DNA in all infected dogs, which were also positive by the real-time PCR. Two days later the ICPOC PCR assay was able to detect only 3/6 infected dogs, which were all positive by the real-time PCR. At days 17 and 19 PI, the ICPOC PCR assay did not detect E. canis DNA in the dogs while the real-time PCR detected all dogs as positive on day 17 PI and two dogs on day 19 PI. In conclusion, the sensitivity of the ICPOC PCR assay was 75% for the acute phase of the disease and 30% for the whole study, suggesting that this ICPOC assay has a potential utility for the diagnosis of acute canine monocytic ehrlichiosis.