Coccidiosis represents a serious threat to the poultry industry, affecting production and causing high morbidity, mortality and significant costs resulting from treatment and prophylaxis. In-feed anticoccidials have been used for decades for managing avian coccidiosis and were very effective until drug resistance emerged. The use of natural remedies has become a promising alternative in combating coccidiosis in chickens. Therefore, the purpose of the present study was to assess the efficiency of a commercial herbal formula (H), as oral liquid preparations, in experimental chicken coccidiosis. Two independent controlled battery experiments (BE1 and BE2) were designed and the product was tested in 3 different formulas (H1, H2 and H3): H1 contained a propylene glycol extract of Allium sativum and Thymus serpyllum; H2 contained Origanum vulgare, Satureja hortensis and Chelidonium majus; and H3 contained Allium sativum, Urtica dioica, Inula helenium, Glycyrrhiza glabra, Rosmarinus officinalis, Chelidonium majus, Thymus serpyllum, Tanacetum vulgare and Coriandrum sativum. Chickens were divided into five groups for each BE as follows: (i) uninfected untreated control (UU1, UU2); (ii) infected untreated control (IU1, IU2); (iii) infected treated with amprolium (ITA1, ITA2); and (iv, v) two experimental groups infected treated with H1 (ITH1) and H2 (ITH2) formulas in the BE1 and with H3 (ITH3-5 and ITH3-10) formula in the BE2. The chickens from infected groups were challenged with 5000 (BE1) and 50,000 (BE2) sporulated oocysts of Eimeria spp. (E. acervulina, E. tenella and E. maxima), respectively. The anticoccidial efficacy was assessed by recording the following: oocysts output (OPG), lesion score (LS), weight gain (WG), feed conversion ratio (FCR) and anticoccidial index (ACI). Additionally, polyphenolics and flavonoids (caffeic-chlorogenic acid, apigenin, kaempferol, luteolin, quercitin, quercitrin) from herb extracts found in H3 formula were determined by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. H1 and H2 reduced the WG, and increased the FCR and OPG compared with controls. H1 reduced the duodenal lesions, whilst H2 reduced the caecal lesions, compared with control. H3 decreased the OPG of Eimeria spp., reduced the total lesion score and improved the zootechnical performance (weight gain and feed conversion ratio). According to ACI value, H1 and H2 had no efficacy on Eimeria spp. infection, but H3 had good to marked anticoccidial effect, the ACI being slightly greater in the group ITH3-5. According to the results of LC-MS/MS, the concentration of polyphenols in H3 formula was the highest, the sum of chlorogenic acid and caffeic acid being 914.9 µg/ml. H3 formula is a promising natural anticoccidial and field trials are recommended in order to validate the obtained data.

Anaplasma platys is a bacterium parasitic in the canine platelets, representing the causative agent of canine cyclic thrombocytopenia, with a worldwide distribution, mainly in tropical countries. The agent has zoonotic potential, being reported in several human clinical cases. The suspected vector is the tick Rhipicephalus sanguineus (sensu lato), widely distributed in animals and humans in the tropical regions of South America, Africa, Asia and Australia, but also in southern Europe. Only few scattered data concerning the epidemiology of A. platys in sub-Saharan Africa are available. The aim of our study was to evaluate the epidemiological status of A. platys in dogs and cats from rural communities in eastern and western Africa, where dogs and their ticks live in close contact with humans. A total of 216 canine and 22 feline blood samples and ticks feeding on them were collected in 2013 and 2014 from eight localities in Ivory Coast and four localities in Kenya. PCR was performed using specific primers targeting a portion of the 16S rRNA gene, followed by sequencing. The main results highlight the high prevalence of A. platys infection in dogs in both Eastern and Western Africa and report it for the first time in Eastern Africa and in Rhipicephalus camicasi. The presence of A. platys DNA in R. camicasi feeding on negative hosts together with the relatively high prevalence of A. platys, correlated with the absence of the probable vector Rhipicephalus sanguineus (s.l.) in Kenyan Island suggest the involvement of other tick species in the transmission of A. platys.

Dirofilaria immitis and Dirofilaria repens are mosquito-borne zoonotic filarioids typically infecting dogs, causing a potentially fatal cardiopulmonary disease and dermatological conditions, respectively. The females are larviparous, releasing the larvae (microfilariae) into the bloodstream, which further develop in mosquito vectors. However, microfilaremia greatly fluctuates during a 24-h period. As the sampling time can greatly influence the accuracy of diagnosis, the aim of the present study was to assess the circadian periodicity of D. immitis and D. repens in naturally co-infected dogs in an endemic area of Romania and to investigate possible differences of periodicity between these two species. Overall, four dogs harbouring natural co-infection with D. immitis and D. repens were selected and sampled every two hours for two consecutive days: two dogs in July 2014 and two in July 2015. At each sampling time, a 0.7 ml blood sample was taken. Modified Knott’s test was performed on 0.5 ml, and the remaining 0.2 ml were used for DNA extraction and molecular amplification, both in single and duplex PCR reactions. Microfilariae of both species were morphologically identified and counted in each collected sample, microfilaremia was calculated, and fluctuation was charted. The dynamics of microfilaremia showed similar patterns for both Dirofilaria species. In all four dogs, D. immitis was present at all sampling times, with several peak values of microfilaremia, of which one was common for all dogs (1 am), while minimum counts occurred between 5 and 9 am. Similarly, for D. repens, one of the peak values was recorded in all dogs at 1 am, while minimum counts (including zero) occurred at 9 and 11 am. Single species-specific PCR reactions were positive for both D. immitis and D. repens in all collected samples, while duplex PCR failed to amplify D. repens DNA in many cases. Both Dirofilaria immitis and D. repens microfilariae are subperiodic, following a similar variation pattern, with peak values of microfilaremia registered during the night in Romania. Duplex PCR fails to identify the infection with D. repens in co-infected dogs when the ratio of microfilaremia is in favour of D. immitis.

Red foxes (Vulpes vulpes) are one of the most widespread wild carnivores in the world, being recognized to harbor and transmit a wide range of vector-borne diseases. Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato are zoonotic tick-borne pathogens causing emerging diseases. Wild animals play an essential role in the transmission of diseases and pathogens maintenance in nature. Epidemiological studies regarding the prevalence of tick-borne pathogens in red foxes are of public health importance, as they may successfully act as a pathogen transmission interface between wildlife, domestic animals and humans. This study included 14 counties from Romania. A total number of 353 red foxes (Vulpes vulpes) were examined. Heart tissue samples were collected during necropsy and stored at −20 °C. Genomic DNA extraction was performed and all samples were examined by polymerase chain reaction (PCR). Specific primers for A. phagocytophilum, A. platys, E. canis and Borrelia burgdorferi s.l. were used. Sequence analysis was performed (Macrogen Europe, Amsterdam) and obtained sequences are available at GenBank™. Out of the 353 samples, 9 (2.55 %; 95 % CI: 1.25–4.96 %) were positive for A. phagocytophilum. Positive animals originated from 5 counties. In total, 5 out of 353 heart tissue samples (1.42 %; 95 % CI: 0.52–3.47 %) collected from red foxes were positive for B. burgdorferi s.l. Red foxes originated from 4 counties. None of the samples were positive for A. platys or E. canis. No co-infection with A. phagocytophilum and B. burgdorferi s.l. was found. This first report of A. phagocytophilum and B. burgdorferi s.l. in red foxes from Romania suggests a limited role of foxes in the maintenance of the two related pathogens, but may represent a potential risk from a public health perspective.

Canine leishmaniasis is a severe, potentially life-threatening, systemic vector-borne disease of dogs caused by protozoan parasites of the genus Leishmania. Romania has been traditionally regarded as a non-endemic country for leishmaniasis with sporadic human disease cases. However, the recent report of an autochthonous canine leishmaniasis case (the first in the last 80 years) suggested the presence of an infection focus in the area of Râmnicu Vâlcea. The present study describes a survey of canine leishmaniasis in this geographical area with comparison to a georeferenced dataset of sand fly distribution based on historical literature records. The study was carried out in Râmnicu Vâlcea and included samples (serum, blood and conjunctival swabs) collected from 80 dogs including client-owned dogs from two local practices and dogs from two public shelters. Serum anti-leishmanial antibodies were assessed by ELISA. All blood and conjunctival samples were assessed by real-time quantitative PCR, targeting the leishmanial kinetoplast minicircle DNA. Three dogs (3.7 %) were seropositive and another four (5.0 %) showed borderline results indicative of exposure or infection. TaqMan PCR was performed for all dogs, on both blood and conjunctival swabs. Seven dogs (8.7 %) were positive by conjunctival swab PCR and one dog (1.2 %) by blood PCR. None of the positive dogs presented clinical signs compatible with canine leishmaniasis. This is the first study evaluating canine leishmaniasis in a dog population in Romania by both highly sensitive PCR and serology. Although the prevalence was relatively low compared to other endemic regions, our results clearly demonstrate the presence of a canine leishmaniasis focus in Romania.

Canine leishmaniasis is a vector-borne zoonotic disease caused by the protozoan parasite Leishmania infantum. In Romania between 1955 and 2013, no cases of human autochthonous visceral leishmaniasis were reported. Data regarding canine leishmaniasis is similarly scarce. Since the first report of clinical autochthonous canine leishmaniasis in 1935, there were only three sporadic reports of positive dogs all without any clinical signs. Our study reports the first clinical case of autochthonous canine leishmaniasis in the last 80 years, stressing the importance of a targeted surveillance of Leishmania infection, as infected dogs act as the primary reservoir for zoonotic visceral leishmaniasis.