Aleksandar Potkonjak

Emerging borreliae – Expanding beyond Lyme borreliosis

Lyme borreliosis (or Lyme disease) has become a virtual household term to the exclusion of other forgotten, emerging or re-emerging borreliae. We review current knowledge regarding these other borreliae, exploring their ecology, epidemiology and pathological potential, for example, for the newly described B. mayonii. These bacteria range from tick-borne, relapsing fever-inducing strains detected in some soft ticks, such as B. mvumii, to those from bat ticks resembling B. turicatae. Some of these emerging pathogens remain unnamed, such as the borrelial strains found in South African penguins and some African cattle ticks. Others, such as B. microti and unnamed Iranian strains, have not been recognised through a lack of discriminatory diagnostic methods. Technical improvements in phylogenetic methods have allowed the differentiation of B. merionesi from other borrelial species that co-circulate in the same region. Furthermore, we discuss members that challenge the existing dogma that Lyme disease-inducing strains are transmitted by hard ticks, whilst the relapsing fever-inducing spirochaetes are transmitted by soft ticks. Controversially, the genus has now been split with Lyme disease-associated members being transferred to Borreliella, whilst the relapsing fever species retain the Borrelia genus name. It took some 60 years for the correlation with clinical presentations now known as Lyme borreliosis to be attributed to their spirochaetal cause. Many of the borreliae discussed here are currently considered exotic curiosities, whilst others, such as B. miyamotoi, are emerging as significant causes of morbidity. To elucidate their role as potential pathogenic agents, we first need to recognise their presence through suitable diagnostic approaches.

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.

Oropharyngeal tularemia in father and son after consumption of under-cooked rabbit meat

Abstract Tularemia has been recognized for more than 10 y in Serbia, since the first epidemic of tularemia occurred in Sokobanja region in 1999. We report 2 cases of oropharyngeal tularemia in a father and son after the consumption of under-cooked rabbit meat. Both presented with fever, unilateral tonsillopharyngitis and cervical lymphadenitis.

Emerging vector-borne diseases - incidence through vectors

Vector-borne diseases use to be a major public health concern only in tropical and subtropical areas, but today they are an emerging threat for the continental and developed countries also. Nowadays, in intercontinental countries, there is a struggle with emerging diseases, which have found their way to appear through vectors. Vector-borne zoonotic diseases occur when vectors, animal hosts, climate conditions, pathogens, and susceptible human population exist at the same time, at the same place. Global climate change is predicted to lead to an increase in vector-borne infectious diseases and disease outbreaks. It could affect the range and population of pathogens, host and vectors, transmission season, etc. Reliable surveillance for diseases that are most likely to emerge is required. Canine vector-borne diseases represent a complex group of diseases including anaplasmosis, babesiosis, bartonellosis, borreliosis, dirofilariosis, ehrlichiosis, and leishmaniosis. Some of these diseases cause serious clinical symptoms in dogs and some of them have a zoonotic potential with an effect to public health. It is expected from veterinarians in coordination with medical doctors to play a fundamental role at primarily prevention and then treatment of vector-borne diseases in dogs. The One Health concept has to be integrated into the struggle against emerging diseases. During a 4-year period, from 2009 to 2013, a total number of 551 dog samples were analyzed for vector-borne diseases (borreliosis, babesiosis, ehrlichiosis, anaplasmosis, dirofilariosis, and leishmaniasis) in routine laboratory work. The analysis was done by serological tests – ELISA for borreliosis, dirofilariosis, and leishmaniasis, modified Knott test for dirofilariosis, and blood smear for babesiosis, ehrlichiosis, and anaplasmosis. This number of samples represented 75% of total number of samples that were sent for analysis for different diseases in dogs. Annually, on average more then half of the samples brought to the laboratory to analysis for different infectious diseases are analyzed for vector-borne diseases. In the region of Vojvodina (northern part of Serbia), the following vector-borne infectious diseases have been found in dogs so far borreliosis, babesiosis, dirofilariosis, leishmaniasis, and anaplasmosis.

Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) in Serbia

Porcine circovirus type 2 (PCV2) is the main causative agent of postweaning multisystemic wasting syndrome (PMWS). To characterize and determine the genetic diversity of PCV2 in the porcine population of Serbia, nucleotide and deduced amino acid sequences of the open reading frame 2 (ORF2) of PCV2 collected from the tissues of pigs that either had died as a result of PMWS or did not exhibit disease symptoms were analyzed. Sequencing and phylogenetic analysis showed considerable diversity among PCV2 ORF2 sequences and the existence of two main PCV2 genotypes, PCV2b and PCV2a, with at least three clusters, 1A/B, 1C and 2D. In order to provide further proof that the 1C strain is circulating in the porcine population, the whole viral genome of one PCV2 isolate was sequenced. Genotyping and phylogenetic analysis using the entire viral genome sequences confirmed that there was a PMWS-associated 1C strain emerging in Serbia. Our analysis also showed that PCV2b is dominant in the porcine population, and that it is exclusively associated with PMWS occurrences in the country. These data constitute a useful basis for further epidemiological studies regarding the heterogeneity of PCV2 strains on the European continent.

Metabolic acclimation to heat stress in farm housed Holstein cows with different body condition scores

The aim of this study was to determine the effects of body condition score to metabolic acclimation in heat stressed Holstein cows. Body condition of cows had no effect on any of the tested parameters during the thermal neutral period, except for the percentage of protein in milk. Heat stress has been demonstrated to have an impact on all the selected parameters, and has been found to be the cause of decreased milk production and quality, increased rectal temperature and respiration rate, decreased glucose, non esterified fatty acids (NEFA) and cholesterol concentration, increased urea and bilirubin concentration, and loss of subcutaneous fat. Obese cows (BCS>4) had less ability of acclimation to heat stress as compared to normal and thin cows (significantly lower production and milk quality, and significantly higher rectal temperature and respiration level in relation to the other groups of cows). Obese cows showed a significantly higher concentration of NEFA and significantly lower glucose during exposure to heat stress. The steady increase in NEFA concentrations and decrease of backfat thickness suggested that fat was used for energy purposes, which significantly increased the heat balance and led to poor acclimation to heat stress in obese cows. Cows with high body condition were at higher risk of developing liver failure and lipidosis (reduced cholesterol and elevated bilirubin concentration) during heat stress, which could be linked with increased concentrations of fatty acids in the blood. This was found with an increased concentration of urea during exposure to heat stress, with significantly higher concentration in obese cows. Using the fat for energy purposes depends on the sensitivity to insulin, which increases during heat stress. Obese cows are naturally less sensitive to insulin and more prone to lipolysis. However, these signs should be the focus of future research.

COMMON FISH SPECIES IN POLYCULTURE WITH CARP AS CYPRINID HERPES VIRUS 3 CARRIERS

Cohabitation studies with common carp were conducted to determine whether the Cyprinid Herpes Virus 3 can infect and establish a productive infection in fish species that according to available data, are not susceptible to this virus. In order to examine if other fish species can contribute to further spreading of the virus, goldfish, silver carp, grass carp, prussian carp and tench were exposed to CyHV-3 through cohabitation with infected carp without clinical symptoms. After this period they cohabitated with naive carp for two weeks and were examined for CyHV-3 by PCR. Our results showed that CyHV-3 was present in the organs of these fish species and also in organs of naive carp after two weeks in cohabitation, suggesting that CyHV-3 may cause latent infection, and also that has a potential to infect a broader host range than it was believed before. Our study adds on better understanding of CyHV-3 transmission not just in its primary host, but also suggests the importance of common fish species in polyculture with carp in the epidemiology of CyHV-3. [Projekat Ministarstva nauke Republike Srbije, br. TR31075 i br. TR-31011]

Study on the effectiveness of topical application of antiseptics in the therapy of digital dermatitis in diary cattle

Digital dermatitis (DD) in cattle is an infectious skin disease of the plantar side of the interdigital space. The causative agents are bacteria from the genus Troponema. Pathomorphologically DD is defined as a combination of ulcerative and proliferative changes which reach down to the dermal papillae and stick out from the ulcerous surface. In order for DD to develop, beside the infective agent, predisposing factors such as wet and unclean floors are required. Treatment of this disorder is possible with the application of antibiotics and/or antiseptic solutions. The aim of this study was to investigate the effectiveness of topical application of antiseptics in the therapy of DD lesions in affected diary cattle. The applied antiseptic solutions were: copper sulfate (8%), zinc sulfate (8%), formalin (8%), and peracetic acid (3%). The treatment was carried out daily during the first seven days, thereon every second day for a total of 30 days. Chlortetracycline was used as the positive control and saline solution was applied as the negative control. By estimation of the presence, size and painfulness of the DD lesions we have established that zinc sulfate had the best curative effect when compared to other antiseptics.

Evaluation of ultrasonography for measuring solar soft tissue thickness as a predictor of sole ulcer formation in Holstein–Friesian dairy cows

The aim of this research was to investigate the correlation between the thickness of solar soft tissue (SST; i.e. the corium and subcutaneous tissue) in early lactation and sole ulcer formation in late lactation. Fifty Holstein-Friesian cows were examined three times: (1) 30 days after calving (locomotion scoring, trimming, claw examination, measurement of SST); (2) 70 days after calving (locomotion scoring); and (3) 180 days after calving (locomotion scoring, trimming, claw examination). SST thickness was measured using an 8 MHz linear probe, at two points (SST1 below the apex of the pedal bone, and SST2 below the flexor tubercle). Mean values for SST1 and SST2 were 3.26 ± 0.43 mm and 4.35 ± 0.46 mm, respectively; the two measures were positively correlated (r=0.95, P<0.001). At 180 days after calving, a sole ulcer was diagnosed in 12/50 cows (24%). Cows with a thinner SST had a higher risk of developing sole ulcers than those with a thicker SST. The optimal cut-off values of SST1 and SST2 in predicting cows with sole ulcers (as calculated by ROC analysis) was ⩽ 3 mm for SST1 and ⩽ 3.9 mm for SST2. The relative risk of a sole ulcer developing, based on those cut-off values was 4.25 (95 CI, 1.77-24.88) for SST1 ⩽ 3 mm and 3.63 (95 CI, 1.18-10.8) for SST2 ⩽ 3.9 mm.

Molecular characterization of Borrelia strains isolated from ticks in Vojvodina

Borrelia burgdorferi sensu lato (s.l.) complex represents a group of different types of spirochete that are present globally, which causes Lyme borreliosis. The total number of types is still not final because new genetic and antigenic isolates are still being described. The clinical picture of Lyme borreliosis in people is polymorphic and is characterized with symptoms similar to flu syndromes together with erythema migrans, rheumatologic, cardiologic and neurological complications. Apart from people, dogs, horses, bovines and sheep can suffer from Lyme borreliosis. In Europe, the most important vector transmitting Lyme disease is the tick Ixodes ricinus. A description of different species of Borrelia in ticks has opened a completely new field of investigating the ecology of Lyme borreliosis. It is especially important to investigate a connection between different types of Borrelia, ticks as vectors and vertebrates as the reservoirs on various geographical localities. The aim of the work is to isolate Borrelia species from the collected ticks I. ricinus from the geographical territory of Vojvodina, and to do a molecular characterization of the isolated strains of Borrelia. A total of 12 tick pools of the type I. ricinus were cultivated in Barbour-Stoenner-Kelly-H medium with additional antibiotics and subcultivated into a modified Kelly-Pettenkofer/Preac-Mursic medium, until a clear culture is reached. For the molecular characterization of isolated strains of Borrelia, molecular methods MluI-LRFP and real-time PCR for hbb gene were used. In this research, out of 12 pools of ticks species I. ricinus 3 strains of Borrelia from the B. burgdorferi s.l. complex were isolated. All three isolates of Borrelia from ticks of the tick species I. ricinus from the territory of Vojvodina were identified as Borrelia afzelii by applying molecular methods (MluI-LRFP and real-time PCR for hbb gene). By applying MluI-LRFP all three isolated strains of B. afzelii were characterized as a subtype Mlal. In this research, like in the previous researches, we have not proved the presence of a pathogenic species Borrelia spielmanii in ticks of the species I. ricinus.