Leptospirosis is a widespread infection among pigs throughout the world. In most cases in Ukraine, only the microscopic agglutination test (MAT) is used for the diagnosis of leptospirosis in animals. In general, during the period of 2001–2019, 2 381 163 samples of blood sera from swine were tested in our country and 85 338 positive reactions were received, which is 3.58% [binomial confidence intervals (BCI), 3.56–3.61%]. It was established that the serovars copenhageni – 33.91% (BCI, 33.59–34.23%), bratislava – 14.14% (BCI, 13.90–14.37%), pomona – 8.58% (BCI, 8.39–8.77%), and tarassovi – 7.12% (BCI, 6.95–7.30%) play a leading role in the aetiological structure of swine leptospirosis. A large number of positive reactions to several serovars was observed – 29.78% (BCI, 29.47–30.09%) of the total number of positive cases. In addition, the article presents data according to a retrospective analysis of the eight serovars circulating among pigs in Ukraine. Thus, during the nineteen year period, there was a decrease in the number of positive reactions to bratislava, pomona, and tarassovi and an increase in the number of positive reactions to copenhageni, polonica, and kabura. Mapping Ukraine’s territory for leptospirosis among pigs was carried out. This allows one to identify zones with a risk of leptospirosis infections among swine. The maps show that the highest incidence rates were identified in the eastern and central parts of Ukraine.
Leptospirosis – common to humans and animals is a dangerous infectious disease that is caused by microbes - leptospires. The disease is accompanied by fever, kidney damage, liver, cardiovascular and nervous system. Leptospirosis in dogs is considered one of the most common diseases. Particularly difficult is the breed with a faulty type of body structure, such as: Neapolitan Mastino, Bulmastiff, English Bulldog, French Bulldog, Boxer, Bologna, Bloodhound, Basset Hound. The disease is most often diagnosed in dogs of hunting breeds, as a result of frequent contact with standing water, as well as in courtyard and stray dogs. Young animals and puppies get sick more often, as they do not have a stable immunity, the hemorrhagic form is more often diagnosed in older dogs. The subject was to study the distribution of leptospirosis among dogs, to establish the seropositivity level and to determine the seroprevalence of the most common of Leptospira serotypes that circulate among this species of animals. For research, an extensive diagnostic series of L. interrogans which includes 20 serovars, and blood serum from dogs that were selected in veterinary clinics in the city of Kyiv, were used as antigen and were transferred to the laboratory of leptospirosis in agricultural animals from the Museum of Microorganisms of the Institute of Veterinary Medicine of the National Academy of Sciences of Ukraine. Studies of blood serum were performed by the microagglutination test (MAT) followed by dark-field microscopy. PMA was placed in 4 dilutions: 1:50, 1: 100, 1: 500 and 1: 2500. According to numerous publications of scientists from different countries of the world, the seroprevalence level of leptospirosis infection among the dogs varies from 39% to 95%. A total of 1831 samples of blood serum were studied in the microscopic agglutination test. As a result of the serological study, 873 animals reacted positively, which is 47.7% of the total number of investigated ones. Analyzing the registered antibody titers, which is most often found titer 1: 100, which is 50.4% of the total number of positive reactions. This indicates the presence of a disease in dogs. Serrogroup Icterohaemorrhagiae is recorded in almost 50% of all positive reactions to leptospirosis and plays a major role in the etiology of the disease. It can be assumed that these dogs had contact with rats or their urine. In turn, the leading for these animals serogroup Canicola was detected in only a third of cases. Other serological groups played a minor role in the etiological structure. Summing up the aforesaid, according to the results of our work, serological prevalence of the pathogenesis of leptospirosis among dogs was determined to be 47.7%. Was detected the circulation of Leptospira`s antibodies in blood serum of these animals. The analysis of the etiological structure of leptospirosis showed that the dominant serogroups were Icterohaemorrhagiae and Canicola. Seven serogroups (Pomona, Sejroe, Grippotyphosa, Australis, Autumnalis, Celledoni, Ballum) were recorded in the range of 1% to 4%. Other serogroups do not have a significant effect on the morbidity of dogs. Keywords: leptospirosis, dogs, etiological structure, serological monitoring, antibody, microscopic agglutination test.
Every year, more than 55,000 people in the world die of rabies, which is confirmed by the data of the WHO expert committee. Up to 40% of victims, from bites sick or suspected of rabies animals are children under the age of 15 years. The source of the rabies agent in 99% of human deaths was dogs. More than 15 million people around the world receive anti-rabies vaccinations after contact with sick or rabies-pets. According to experts, this prevents 327 thousand deaths from rabies a year. The susceptibility to skeletal infection of various species of animals, involvement in the epizootic chain, in addition to wildlife, also in domestic and farm animals, has become an extremely high risk for humans, and the lack of means for treating rabies - determine its special place among all contagious diseases. In the current situation, a rabie must be viewed as an international rather than a local or national problem, so it describes the disease as a global scale noseau [17]. After all, according to the statistics of the International Office of Epizootics (MEB) of the governing body of the World Organization for Animal Health (WHO) cases of rabies recorded in more than 150 countries of the world. Rabies free are only the countries of Oceania and the United Kingdom, and in other countries the sporadic cases of this deadly disease are recorded. In Europe, this infection in the 50s has become epizootic. The "eradication" of the rabies virus in different countries of Europe during 2008-2015 was conducted in accordance with the developed and implemented Program, where the best results were received in Germany and Switzerland, in these countries the oral immunization of wild carnivores is used (without restrictions on funding). At the end of the last century, the epicenter of the rabies began to move from Eastern Europe to the territory of Poland (2001-2002), Croatia (2003) and then to the east - to the Russian Federation, the Republic of Belarus, Latvia and Ukraine. The conducted monitoring of the rabies epizootology have shown that the entire territory of Ukraine is a zone of stable disadvantage of this disease. The peak of epizootics in Ukraine, over the past 65 years, has fallen to 2007 (2393 cases). Since 2008 there were registered from one to two thousand cases of rabies. In 2017 there were registered 1356 cases of animals that were sick on rabies, despite the fact those more than 4.2 million anti-rabies vaccinations of domestic animals. The analysis of the situation shows that from year to year in Ukraine new natural fires of rabies are formed and new types of reservoir animals appear, which leads to an increase in the number of outbreaks of this disease. The main pledge of successful prevention of rabies around the world is the use of effective anti-rabies vaccines. Despite the regular implementation of planned antiepizootic measures in the southern region of Ukraine (including in the Skadovsk district), there is a tendency to spread the rabies. From year to year, a significant number of rabies in wildlife, domestic and farm animals are recorded on this territory, which is a real threat to the occurrence of the disease among humans. Investigations of the territory of the Skadovsky district of the Kherson region during the period from 2013 to 2017 showed that 25 cases of rabies were recorded in 15 settlements. To the zone with high tenseness of the epizootic situation, five settlements can be attributed, in which 15 cases of rabies were registered, namely in the villages: Krasnoye and Mykolaivka for four outbreaks, Tarasivtsi three, Ptakovtsi and Blagodatnyi for two. To the zone with low tensity epizootic situation still belongs to 10 settlements, where one case of rabies is registered. If in 2013 and 2016 there were three rabies outbreaks in four settlements, in 2014 - five cases in 5 villages, and in 2015 - 4 cases in 4 settlements, then in 2017 - 10 outbreaks communicable infection in eight villages of the district. The peak of this epizootic occurred in 2017. The conducted monitoring showed that the skeletal infection was registered in 38.5% of the Skadovsky district (in 15 out of 39 available settlements). The cases of rabies in different years are interrelated, since repeated outbreaks of this disease are recorded in previously unsuccessful places (Nikolaevka, Tarasivka, Red and Ptahovka), indicating the presence of a constant source and reservoir of communicable infection in this area. It is alarming that in 2017 cases of rabies were first recorded in 6 settlements (Blagodatnoy, Grushivtsi, Oleksandrivka, Petropavlivtsi, Zeleny and Mikhailovna), which testifies to the uncontrolled epizootic situation and the further spread of this infection in the Skadovsky district. Thus, during the last 5 years, the activity of manifestation of the epizootic process on the territory of Skadovsky area was not marked by stability, there were observed two periods of lifting epizootics of rabies - in 2014 and 2017. The permanent disadvantages of certain areas of Skadovsky district can be explained, first of all, by the presence of natural cells of the common infection, especially in the steppe part where there are bushes, empty garbage and garbage. Such conditions ensure the existence of a wide variety of wild populations (wolves, red foxes), homeless dogs and stray cats, which promotes the spread of rabies epizootics in this area. Not the timely destruction of wild animals, the catching of homeless animals that are dangerous to humans, their sterilization, the creation of shelters and proper conditions for maintenance, and in the last three years, not even the holding of oral inoculations of wild animals, has led to the emergence of new anthropological cells feline infections in this region. Monitoring surveys of all reported cases of rabies in Skadovsk district for 2013-2017 showed that in this area, the source of the rabies was different species of animals - wild, domestic and agricultural. According to the observations of many scholars, rabies does not belong to seasonal diseases, but in Skadovsk district 20 were recorded in the winter and spring months, namely: 4 outbreaks of this infection were registered in November, December, February and March, 1 in January and 2 in October. From April to August - 5 outbreaks of rabies. Seasonal cases of rabies coincide with the period of racing of foxes. In the summer, the number of diseases is minimal, because the foxes are busy raising babies, and therefore their mobility is limited. In the autumn, due to young individuals, the population density increases, and accordingly there is a proliferation and new growth of this epizootic. By increasing the population of wolves and foxes, the number of rabies cases among stray dogs and stray cats increases as a result of their contacts. In the disadvantaged areas of the district, over the past 5 years, 27 cases were diagnosed with rabies animals. Monitoring studies have shown that in 2013 the rabies virus is allocated from 4 diseased animals, in 2014 from 5, in 2015 - 4, in 2016 - 3 and in 2017 - 11 diseased rabies animals. Significant increase in the morbidity rate of animals was noted in 2017. It is known that different species of animals are susceptible to the rabies virus [1, 2, 6–9, 16, 34]. The conducted studies showed that in the Skadovsky area, the circulation of the virus of cutaneous infection is possible among different animals, because the source of the pathogen was six of their species: foxes, wolves, dogs, cats, large and small cattle. In the structure of the morbidity of animals in rabies, foxes and cats occupy the leading place with 33.3%, dogs - 14.8%, wolves and cattle - by 7.5% and DRH - 3.7%. Of the total number of ill in 41% of cases, the source of the rabies virus were wild animals, and 48% were domesticated. The analysis showed that rabies in Skadovsk district was more often registered among domestic animals - 48% (9 cats and 4 dogs). Among wild animals, patients with rabies were - 41% (11 heads, of which 9 foxes and 2 wolves), and agricultural - 11% (cow, calf and goat). Thus, the statistical data of this region confirm that the source and reservoir of the rabies agent are wild (foxes) and domestic predatory animals (dogs and cats) belonging to the class of mammals. The epizootic situation in the Skadovsk district from rabies is not catastrophic, but in recent years requires more attention and strengthening measures to combat this problem, because in 2016, the density of fox in the district was 3 heads for 1000 hectares of land; in the norm for example - 0,5 - 1 a goal for 1000 hectares of land. Rating of wolves has not been conducted here, although cases of rabies among this species of animals, for the last 5 years, were recorded twice. The increased morbidity of dogs, cats and farm animals for scarcity is a sign of epizootic malaise among wildlife. Contributes to the complication of the situation of growth in settlements of the number of homeless dogs and cats, incomplete coverage of preventive vaccinations of domestic animals, violation of the rules for keeping domestic animals by their owners. All this is a prerequisite for the formation of city-type rabies cells, which we observe in Sadovsky district of the Kherson region. In connection with the deterioration of the epizootic situation from rabies, the threat of the onset and spread of this infection among the population increases. Given that the Skadovsk district of the Kherson region is in the resort zone, and a significant number of Ukrainian and foreign citizens may come to rest on the sea, they must be aware of the epizootic rabies situation in the area in order to protect themselves from the deadly infection. Key words: contagious infection, dogs, cats, red foxes, wolves, epizootic well-being, morbidity, seasonal manifestations.
ObjectiveTo introduce the method of molecular genotyping (MLVA) to determine the genotype of field isolates of leptospira.IntroductionLeptospirosis (ictherohemoglobinuria, Leptospirosis biliousness) is a natural focal and zoonotic infectious disease dangerous for humans and farm animals. It is important to identify specific leptospira strains isolated from rodents or sick and suspicious animals by the serotype or genotype. In comparison with serotyping using micro agglutination test (MAT), molecular genotyping makes it possible to accurately identify a specific pathogen strain. The genetic classification now becomes more significant than the phenotypic classification.MethodsSpecific oligonucleotide primers, which flank fragments of the genome locus of pathogenic leptospira varies in terms of the number of tandem repeats VNTR-4, -7, -10 specific for L.interrogans, L.kirschneri, and L.borgpetersenii were used. The amplification products were detected using agar gel electrophoresis with the following identification of the fragment length with a molecular weight marker and comparison with the collection of VNTR profiles of the strains described in the literature.ResultsIt was established that the method of leptospira molecular genotyping by determining the number of variable tandem repeats of a locus (VNTR-variable number tandem repeats analysis) is suitable for molecular epizootology studies in Ukraine. The advantages of the method are the simplicity of performance and availability for diagnostic and research laboratories in Ukraine compared to other pathogen genome sequencing based genotyping methods, in particular Multilocus sequence typing (MLST) or Multispacer Sequence Typing (MST), which require complex equipment and operating conditions. The reference strain of Leptospira M20 serotype Copengageni serogroup Icterohaemorrhagiae from the NAAS IVM collection of was studied and its VNTR profile was identified with the genotype of the strain Fiocruz L1-130 that is described in the literature as a serotype of Copengageni serogroup Icterohaemorrhagiae. The genotype of the leptospira field isolate obtained from a rat in Lviv Oblast of Ukraine was specified and its identity was established in the aforementioned genotype. The obtained data support the prospects of using MLVA genotyping method to study the distribution of different genotypes of leptospira. The research will continue to study the specificities of molecular epizootology of leptospirosis in Ukraine.ConclusionsThe method of leptospira molecular genotyping by multilocus analysis of the number of variable tandem repeats has been tested in the Leptospirosis Research Laboratory in collaboration with the Museum of Microorganisms at the National Academy of Sciences, the Ukraine Institute of Veterinary Medicine, the ELISA and PCR Research Laboratory, and the Bila Tserkva National Agrarian University. The genotype of the reference strain has been correlated with its serological profile; identification of the genotype of the field isolate pathogenic leptospira has been completed. The tested method is planned to be implemented in surveillance and control over leptospirosis spreading in Ukraine, and aimed to help in development and improvement of leptospirosis vaccine formulations. Additionally, method of Multiple-Locus Variable number tandem repeat Analysis will be used for molecular epidemiology research in Ukraine.ReferencesSalaün L, Mérien F, Gurianova S, Baranton G, Picardeau M. Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. J Clin Microbiol. 2006;44(11):3954-3962. doi:10.1128/JCM.00336-06.Caimi K, Repetto SA, Varni V, Ruybal P. Infection , Genetics and Evolution Leptospira species molecular epidemiology in the genomic era. Infect Genet Evol. 2017;54(July):478-485. doi:10.1016/j.meegid.2017.08.013.Ayral F, Zilber AL, Bicout DJ, Kodjo A, Artois M, Djelouadji Z. Distribution of leptospira interrogans by multispacer sequence typing in urban Norway rats (Rattus norvegicus): A survey in France in 2011-2013. PLoS One. 2015;10(10):1-14. doi:10.1371/journal.pone.0139604.
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