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Newcastle disease is one of the most important and highly contagious disease for poultry. The causative agent Avian avulavirus 1 belongs to genus Avulavirus, Family Paramyxoviridae, order Mononegavirales. There are two evolutionary related groups AAvV-1 – class I and class II. The classification is based on complete genome analysis. Class I and class II being further divided in genotypes. However, the isolation and identification of novel avulaviruses of varying pathogenicity and genotypes from a wide range of hosts suggests a con-tinuous virus evolution with subsequent implications for disease spread. Subsequent to evolution, the emer-gence of novel genotypes and sub-genotypes are now increasing over a period of time. Despite the introduction of a broad vaccination program in industrial poultry farms in Bulgaria, the birds in the backyard farms have not been vaccinated and the disease has become endemic. Almost every year there is at least one outbreak in poultry or wild birds. The earlier AAvV-1 isolates, from 1959 to 1998, belong to many different genetic lines: 2, 3b, 3c, 4 and 5b. The last AAvV-1 isolates (from 2004 to 2013) from chickens belong to genetic line 5d and wild bird isolates belongs to 1, 2, 4b and 6. The first appearance of genetic line 5a is in the beginning of 2013. All this necessitates in-depth studies of AAvV-1 in the population of both wild and synanthropic birds and vaccinated hens. This study was conducted to ascertain the genetics of the circulating AAvV-1 that causes disease outbreaks in 2016 and 2017., {"references":["Hadzhiev G. (1973). \"Prouchvania varhu nqkoi svoistva na shtamovete na virusa na psevdochumata po pticite, izolirani v Bulgaria\". Disertacionen trud za prisujdane na obrazovatelna I nauchna stepen \"doctor\", NDRIVMI, Sofia. pp. 1–214.","Aldous E.W., Mynn J.K., Banks J., Alexander D.J. (2003). A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene. Avian Pathol. 32(3), 239–56.","Alexander, D. J., (1997). Newcastle disease and other Paramyxoviridae infections. In B. W. Calnek, H. J. Barnes, C. W. Beard, L. McDougald, and J. Y. M. Saif (ed.), Diseases of poultry, 10th ed. Iowa State University, Ames, Iowa. pp. 541–569.","Alexander, D. J. (1998). Newcastle disease virus and other avian paramyxoviruses. In D. E. Swayne, J. R. Glisson, M. W. Jackwood, J. E. Pearson, and W. M. Reed (ed.), A laboratory manual for the isolation and identification of avian pathogens, 4th ed. American Association of Avian Pathologists, Kennett Square, Pa. pp. 156–168.","Alexander, D. J., Manvell R. J., Banks J., Collins M.S., Parsons G., Cox B., Frost K.M., Speidel E.C., Ashman S., Aldous E.W. (1999). Experimental assessment of the pathogenicity of the New-castle disease viruses from outbreaks in Great Britain in 1997 for chickens and turkeys, and the protection afforded by vaccination. Avian Pathology, 28, 501–512.","Czeglédi, A., Ujvári D., Somogyi E., Wehmann E., Werner O., Lomniczi B. (2006). Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary impli-cations. Virus Res.120 (1–2), 36–48.","Damena D., Fusaro A., Sombo M., Belaineh R., Heidari A., Kebede A., Kidane M., Chaka H., (2016). Characterization of Newcastle disease virus isolates obtained from outbreak cases in com-mercial chickens and wild pigeons in Ethiopia. Springer Plus. 5, 476.","Dimitrov K. M., Ramey A. M., Qiu X., Bahl J., Afonso C. L., (2016). Temporal, geographic, and host distribution of avian paramyxovirus 1 (Newcastle disease virus). Infect. Genet. Evol. 39, 22–34.","Herczeg J., Wehmann E., Bragg R. R., Travassos Dias P. M., Hadjiev G., Werner O., Lomniczi B. (1999). Two novel genetic groups (VIIb and VIII) responsible for recent Newcastle disease out-breaks in Southern Africa, one (VIIb) of which reached Southern Europe. Arch. Virol. 144, 2087–2099.","Herczeg J., Pascucci S., Massi P., Luini M., Selli L., Capua I., Lomniczi B. (2001). A longitudinal study of velogenic Newcastle disease virus genotypes isolated in Italy between 1960 and 2000. Avian Pathology, 30, 163–168.","International Committee on Taxonomy of Viruses (ICTV). https://talk.ictvonline.org/taxonomy/","Kim L. M., King D. J., Curry P. E., Suarez D. L., Swayne D. E., Stallknecht D. E., Slemons R. D., Pedersen J. C., Senne D. A., Winker K., Afonso C. L. (2007). Phylogenetic diversity among low–virulence newcastle disease viruses from waterfowl and shorebirds and comparison of genotype distributions to those of poultry–origin isolates. J Virol. 81(22), 12641–53.","Lomniczi B., Wehmann E., Herczeg J., Ballagi–Pordany A., Kaleta E. F., Werner O., Meulemans D., Jorgensen P. H., Mante A. P., Gielkens A. L. J., Damoser J. (1998). Newcastle disease outbreaks in recent years in Western Europe were caused by an old (VI) and a novel genotype (VII). Archives of Virology, 143, 49–64.","Wise M. G., Suarez D. L., Seal B. S., Pedersen J. C., Senne D. A., King D. J., Kapczynski D. R., Spackman E. (2004). Development of a Real–Time Reverse–Transcription PCR for Detection of Newcastle Disease Virus RNA in Clinical Samples. J Clin Microbiol.42(1), 329–338.","Miller P. J., Kim L. M., Ip H. S., Afonso C. L. (2009). Evolutionary dynamics of Newcastle disease virus. Virology.15, 391(1), 64–72.","OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2016, chapter 2.3.1.14. http://www.oie.int/international–standard–setting/terrestrial–manual/.","Courtney S. C., Susta L., Gomez D., Hines N. L., Pedersen J. C., Brown C. C., Miller P. J., Afonso C. L. (2013). Highly Divergent Virulent Isolates of Newcastle Disease Virus from the Do-minican Republic Are Members of a New Genotype That May Have Evolved Unnoticed for Over 2 Decades. J Clin Microbiol.51(2), 508–517.","Haji–Abdolvahab H., Ghalyanchilangeroudi A., Bahonar A., Ghafouri S.A., Marandi M.V., Mehrabadi M.H.F., Tehrani F. (2019). Prevalence of avian influenza, Newcastle disease, and infectious bronchitis viruses in broiler flocks infected with multifactorial respiratory diseases in Iran, 2015–2016. Tropical Animal Health and Production, 51, 689–695. doi: 10.1007/s11250-018-1743-z.","Kolakofsky D., Roux L., Garcin D., Ruigrok R.W. (2005). Paramyxovirus mRNA editing, the 'rule of six' and error catastrophe: a hypothesis. Journal of General Virology, 86, 1869–1877. doi: 10.1099/vir.0.80986–0.","Alexander D.J., Aldous E.W., Fuller C.M. (2012). The long view: a selective review of 40 years of Newcastle disease research. Avian Pathology, 41, 329–335. doi: 10.1080/03079457.2012.697991","Ramey A.M., Reeves A.B., Ogawa H., Ip H.S., Imai K., Bui V.N., Yamaguchi E., Silko N.Y., Afonso, C.L. (2013). Genetic diversity and mutation of avian paramyxovirus serotype 1 (Newcastle disease virus) in wild birds and evidence for intercontinental spread. Archives of Virology, 158, 2495–2503. doi: 10.1007/s00705–013–1761–0.","Shabbir M.Z., Akhtar S., Tang Y., Yaqub T., Ahmad A., Mustafa G., Alam M.A., Santhakumar D., Nair V. (2016). Infectivity of wild bird origin avian Paramyxovirus serotype 1 and vaccine effec-tiveness in chickens. Journal of General Virology, 97, 3161–3173. doi: 10.1099/jgv.0.000618.","Brown V.R., Bevins S.N. (2017). A review of virulent Newcastle disease viruses in the United States and the role of wild birds in viral persistence and spread. Veterinary Research, 48, 68. doi: 10.1186/s13567–017–0475–9.","Dodovski A., Cvetkovikj I., Krstevski K., Naletoski I., Savić V. (2017). Characterization and epi-demiology of pigeon paramyxovirus type–1 viruses (PPMV–1) isolated in Macedonia. Avian Dis-eases, 61, 146–152. doi: 10.1637/11517–101816–Reg.1.","Qiu X., Meng C., Zhan Y., Yu S., Li S., Ren T., Yuan W., Xu S., Sun Y., Tan L., Song C. (2017). Phylogenetic, antigenic and biological characterization of pigeon paramyxovirus type 1 circulating in China. Virology Journal, 14, 186. doi: 10.1186/s12985–017–0857–7.","El Naggar R.F., Rohaim M.A., Bazid A.H., Ahmed K.A., Hussein H.A., Munir M. (2018). Biologi-cal characterization of wild–bird–origin avian avulavirus 1 and efficacy of currently applied vac-cines against potential infection in commercial poultry. Archives of Virology, 163, 2743–2755. doi: 10.1007/s00705–018–3916–5.","Aziz–ul–Rahman, Munir M., Shabbir M.Z. (2018). Comparative evolutionary and phylogenomic analysis of avian avulaviruses 1–20. Molecular Phylogenetics and Evolution, 127, 931–951. doi: 10.1016/j.ympev.2018.06.040.","Miller P.J., Haddas R., Simanov L., Lublin A., Rehmani S.F., Wajid A., Bibi T., Khan T.A., Yaqub T., Setiyaningsih S., Afonso C.L. (2015). Identification of new sub–genotypes of virulent Newcastle disease virus with potential panzootic features. Infection, Genetics and Evolution, 29, 216–229. doi: 10.1016/j.meegid.2014.10.032.","Fan W., Wang Y., Wang S., Cheng Z., Guo H., Zhao X., Liu J. (2017). Virulence in Newcastle disease virus: a genotyping and molecular evolution spectrum perspective. Research in Veterinary Science, 111, 49–54. doi: 10.1016/j.rvsc.2016.12.001.","Dimitrov K.M., Abolnik C., Afonso C.L., Albina E., Bahl J., Berg M., Briand F.X., Brown I.H., Choi K.S., Chvala I., Diel D.G. (2019a). Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus. Infection, Genetics and Evolution, 74: 103917. doi: 10.1016/j.meegid.2019.103917.","Liang R., Cao D.J., Li J.Q., Chen J., Guo X., Zhuang F.F., Duan M.X. (2002). Newcastle disease outbreaks in western China were caused by the genotypes VIIa and VIII. Veterinary Microbiology, 87, 193–203. doi: 10.1016/S0378–1135(02)00050–0.","Abolnik C., Mubamba C., Wandrag D.B., Horner R., Gummow B., Dautu G., Bisschop S.P. (2018). Tracing the origins of genotype VII h Newcastle disease in Southern Africa. Transboundary and Emerging Diseases, 65, e393–e403. doi: 10.1111/tbed.12771.","Dimitrov K.M., Ferreira H.L., Pantin–Jackwood M.J., Taylor T.L., Goraichuk I.V., Crossley B.M., Killian M.L., Bergeson N.H., Torchetti M.K., Afonso C.L., Suarez D.L. (2019b). Pathogenicity and transmission of virulent Newcastle disease virus from the 2018–2019 California outbreak and re-lated viruses in young and adult chickens. Virology, 531, 203–218. doi: 10.1016/j.vi-rol.2019.03.010.","Amarasinghe G.K., Ayllón M.A., Bào Y., Basler C.F., Bavari S., Blasdell K.R., Briese T., Brown P.A., Bukreyev A., Balkema–Buschmann A., Buchholz U.J. (2019). Taxonomy of the order Mo-nonegavirales: update 2019. Archives of Virology, 164, 1967–1980. doi: 10.1007/s00705–019–04247–4."]}

The aim of the present study was to determine the microbiological status of the mammary gland in lactating sheeps of the Lacon breed. For this purpose, milk samples from purebred animals, rais��d in sheep farm located in central region of Bulgaria, were collected. The results show that in 46.6% of the tested milk samples from the farm, were not isolated any pathogenic microorganisms. From the positive samples, S. xylosus was most often found, in most cases in combination with the non-pathogenic streptococcus L. lactis ssp. lactis. Only in one of the samples a combination of two staphylococcal species was found ��� S. epidermidis and S. xylosus, with the presence of L. lactis ssp. lactis. The number of samples with isolated bacteria with pathogenic potential was 16 (53.3%). Only in seven of them (23.3% of all examined) the amount of detected staphylococci is over 104 CFU/ml. No Gram-negative bacteria were isolated from any of the milk samples. The sensitivity of the isolated bacteria from milk samples to antibiotics from different groups is significant. Resistance has only been established to Colistin, Kanamycin, Amikacin and Gentamicin. The mean values of somatic cells in infected and uninfected halves were 1361058 �� 361307.34 and 171000 �� 36586.04, respectively., {"references":["Ahmad Z., S. Agarwal, A. S. Narwal, V. K. Deshwal. (2013). Comparative study of antibiotics re-sistance of coagulase negative staphylococci. Developmental Microbiology and Molecular Biology, 4, 29–33","Ariznabarreta A., Gonzalo, C., San Primitivo, F. (2002). Microbiological quality and somatic cell count of ewe milk with special reference to staphylococci. J. Dairy Sci. 85, 1370–1375","Bauer A. W., W. M. Kirby, J. C. Cherris, M. Truck. (1966). Antibiotic susceptibility testing by a standardized single disk method. The Am. J. of Clin. Pathol. 45 (4), 493–496.","Bergonier D., X. Berthelot. (2003). New advances in epizootiology and control of ewe mastitis. Livest. Prod. Sci. 79, 1–16.","Boutinaud M., Jammes, H. (2002). Potential uses of milk epithelial cells: a review. Reprod. Nutr. Dev. 42, 133–147","Brugère-Picoux J. (2008): Ovine listeriosis. Small Ruminant Research, 76, 12–20","Contreras A., D. Sierra, J.C. Corrales, J.C. Marco, M.J. Paape, C. Gonzalo. (2007). Mastitis in small ruminants. Small Ruminant Research 68, 145–153.","Fotou K. A.Tzora, C. H. Voidarou, A. Alexopoulos, E. Plessas, I. Avgeris, E. Bezirtzoglou, K. Akrida-Demertzi, P. G. Demertzis. (2011). Isolation of microbial pathogens of subclinical mastitis from raw sheep's milk of Epirus (Greece) and their role in its hygiene. Anaerobe, 315–319.","Gelasakis A.I., V.S. Mavrogianni, I.G. Petridis, N.G.C. Vasileiou, G.C. Fthenakis. (2015). Mastitis in sheep – The last 10 years and the future of research. Small Ruminant Research 88, 119–125","Holt J. G., N. R. Krieg, P. H. A. Sneart, J. T. Staley, S. T. (1994). Williams. Bergey's manual of determinative bacteriology, Ninth edition. Williams & Wilkins, 209–210, 538–562","Hristov K., T. Popova, R. Pepovich and B. Nikolov. (2016). Characterization of Microbial Causa-tive Agents of Subclinical Mastitis in Goats in Bulgaria. Int. J. Curr. Microbiol. App. Sci. 5 (8), 316–323.","Juste R.A., E. Minguijón, J. Arranz, M. Fuertes, I. Beltrán de Heredia. (2009). Lamb mortality in an outbreak of Yersinia pseudotuberculosis mastitis as a collateral effect of colostrums feeding for Lentivirus-control. Small Ruminant Research 86, 46–51.","Leitner G., U. Merin, Y. Lavi, A. Egber, N. (2007). Silanikove Aetiology of intramammary infection and its effect on milk composition in goat flocks J. Dairy Research 74, 186–193.","Marogna G., S. Rolesu, S. Lollai, S. Tola, G. Leori. (2010): Clinical findings in sheep farms affected by recurrent bacterial mastitis. Small Ruminant Research, 88, 119–125.","Martins K. B., P. Y. Faccioli, M. F. Bonesso, S. Fernandes, A. A. Oliveira, A. Dantas, L. F. Zafalon, M. de Lourdes, R. S. Cunha. (2016). Characteristics of resistance and virulence factors in different species of coagulase-negative staphylococci isolated from milk of healthy sheep and animals with subclinical mastitis. J. Dairy Sci., 100, 2184–2195","Mavrogianni V.S., P.I. Menzies, I.A. Fragkou, G.C. Fthenakis. (2011) Principles of mastitis treat-ment in sheep and goats. Vet. Clin. North Am. Food Anim. Pract., 27, 115–120.","Nebbia P., P. Robino, S. Zoppi, D. De Meneghi. (2006). Detection and excretion pattern of Myco-bacterium avium subspecies paratuberculosis in milk of asymptomatic sheep and goats by Nested-PCR. Small Ruminant Research, 66, 116–120.","Queiroga MC. (2017). Prevalence and aetiology of sheep mastitis in Alentejo region of Portugal. Small Ruminant Research, 153, 123–130.","Rodrigues M. X., S. F. Lima, C. H. S. G. Canniatti-Brazaca, R. C. Bicalho. (2016). The Lactococcus genus as a potential emerging mastitis pathogen group: A report on an outbreak investigation. Jour-nal of Dairy Science, 99, 12, 9864–9874.","Saratsis P., L. Leontides, A. Tzora, C. Alexopoulos, G. C. Fthenakis. (1998). Incidence risk and aetiology of mammary abnormalities in dry ewes in 10 flocks in southern Greece. Prev Vet Med., 37(1–4), 173–83.","Spanu C., Y. M. Berger, D. L. Thomas, P. L. Ruegg. (2011). Impact of intramammary antimicrobial dry treatment and teat sanitation on somatic cell count and intramammary infection in dairy ewes. Small Ruminant Research, 97, 139–145.","Sylejmani R., A. Robaj, A. Hamidi. (2015). Antimicrobial susceptibility of staphylococci isolated from clinical mastitis in dairy cows in two regions of kosovo. Albanian j. agric. sci., 14(4), 404–407.","Thomson K, M. Rantala, M. Hautala, S. Pyӧrälä, L. Kaartinen. (2008). Cross-sectional prospec-tivesurvey to study indication–based usage of antimicrobials in animals: Results of use incattle. Veterinary Research, 15.","Tvarožková K., V. Tančin, M. Uhrinčať, L. Hleba, L. Mačuhová. (2020). Mastitis pathogens and somatic cell count in ewes milk. Potravinarstvo Slovak Journal of Food Sciences, 14, 164–169.","Vasileiou N.G.C., P.J. Cripps, K.S. Ioannidi, D.C. Chatzopoulos, D.A. Gougoulis, S. Sarrou, D.C. Orfanou, A.P. Politis, T. Calvo Gonzalez-Valerio, S. Argyros, V.S. Mavrogianni, E. Petinaki, G.C. Fthenakis. (2018). Extensive countrywide field investigation of subclinical mastitis in sheep in Greece. J. Dairy Sci. 101, 7297–7310."]}

Vector-borne diseases pose a serious threat to public health and animal husbandry and require special attention. Knowledge of the ecology of different insect species acting as vectors of multiple infectious agents will be crucial for the development and implementation of appropriate and effective vector control strategies to reduce the impact of the diseases they transmit. However, the main limitation is that morphological methods for identifying insect species are time consuming and require taxonomic expertise. Inaccurate identification of vector species can have a significant impact on attempts to control their populations. One of the remarkable achievements of modern biology and molecular diagnostic methods is the development of accurate and reliable technologies for rapid screening of DNA sequence variations. Molecular identification and DNA barcoding is a relatively new taxonomic method that can be used to identify a species, including an unknown one. Closely related or unknown species can be differentiated. DNA barcoding is a standardized approach to identifying plants and animals through minimal DNA sequences called DNA bar-codes., {"references":["Jinbo U., Kato T., Ito M. (2011). Current progress in DNA barcoding and future implications for entomology. Entomol Sci, 14:107–124","Sirivanakarn, S. (1977). A revision of the subgenus Lophoceraomyia of the genus Culex in the Ori-ental region (Diptera: Culicidae). Contrib Am Entomol Inst (Ann Arbor), 13:1–245.","Hebert, P. D., Cywinska A., Ball S. L., de Waard J. R. (2003). Biological identifications through DNA barcodes. Proc Biol Sci, 270:313–321.","Hebert, P. D., Ratnasingham S., De Waard J. R. (2003). Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Biol Sci, 270(Suppl 1): S96–S99.","Rueanghiran, C., Apiwathnasorn C., Sangthong P., Samung Y., Ruangsittichai J. (2011). Utility of a set of conserved mitochondrial cytochrome oxidase subunit I gene primers for Mansonia annulata identification. Southeast Asian J Trop Med Public Health, 42:1381–1387","Paz, A., Crawford A. J. (2012). Molecular-based rapid inventories of sympatric diversity: a com-parison of DNA barcode clustering methods applied to geography-based vs clade-based sampling of amphibians. J Biosci, 37:887–896.","Merget, B., Koetschan C., Hackl T., Forster F., Dandekar T., Muller T., Schultz J., Wolf M. (2012). The ITS2 Database. J Vis Exp, 61:pii: 3806","Sevilla, R. G., Diez A., Norn M., Mouchel O., Jerome M., Verrezagnis V., Van Pelt H., Favrery, L., Krey G., Bautista J.M. (2007). Primers and polymerase chain reaction conditions for DNA barcod-ing teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes. Mol Ecol Notes, 7:730–734.","Shen, Y. Y., Chen X., Murphy R. W. (2013). Assessing DNA barcoding as a tool for species identi-fication and data quality control. PLoS ONE, 8:e57125.","Vences, M., Thomas M., van der Meijden A., Chiari Y., Vieites D. R. (2005). Comparative perfor-mance of the 16S rRNA gene in DNA barcoding of amphibians. Front Zool, 2:5.","Chu, K. H., Li C., Qi J. (2006). Ribosomal RNA as molecular barcodes: a simple correlation anal-ysis without sequence alignment. Bioinformatics, 22:1690–1701.","Rach, J., Desalle R., Sarkar I. N., Schierwater B., Hadrys H. (2008). Character-based DNA barcod-ing allows discrimination of genera, species and populations in Odonata. Proc Biol Sci, 275:237–247.","Webster, B. L., Emery A. M., Webster J. P., Gouvras A., Garba A., Diaw O., Seye M. M., Tchuente L. A. T., Simoonga C., Mwanga J. (2012). Genetic diversity within Schistosoma haematobium: DNA barcoding reveals two distinct groups. PLoS Negl Trop Dis, 6:e1882","Laurito, M., de Oliveira T. M., Almiron W. R., Sallum M. A. M. (2013). COI barcode versus mor-phological identification of Culex (Culex) (Diptera: Culicidae) species: a case study using samples from Argentina and Brazil. Mem Inst Oswaldo Cruz, 108:110–122","Khamis, F. M., Masiga D. K., Mohamed S. A., Salifu D., de Meyer M., Ekesi S. (2012). Taxonomic identity of the invasive fruit fly pest. Bactrocera invadens: concordance in morphometry and DNA barcoding. PLoS One, 7:e44862","Roy, S., Tyagi A., Shukla V., Kumar A., Singh U. M., Chaudhary L. B., Datt B., Bag S. K., Singh P. K., Nair N. K. (2010). Universal plant DNA barcode loci may not work in complex groups: a case study with Indian Berberis species. PLoS ONE, 5:e13674","Schoch, C. L., Seifert K. A., Huhndorf S., Robert V., Spouge J. L., Levesque C. A., Chen W., Bol-chacova E., Voigt K., Crous P. W. (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A 2012, 109:6241–6246.","Wang, G., Li C., Guo X., Xing D., Dong Y., Wang Z., Zhang Y., Liu M., Zheng Z., Zhang H. (2012). Identifying the main mosquito species in China based on DNA barcoding. PLoS ONE, 7:e47051","Kumar, N. P., Rajavel A. R., Natarajan R., Jambulingam P. (2007). DNA barcodes can distinguish species of Indian mosquitoes (Diptera: Culicidae). J Med Entomol 2007, 44:1–7.","Bourke, B. P., Oliveira T. P., Suesdek L., Bergo E. S., Sallum M. (2013). A multi-locus approach to barcoding in the Anopheles strodei subgroup (Diptera: Culicidae). Parasit Vectors, 6:111.","Rubinoff, D. (2006). DNA barcoding evolves into the familiar. Conser Biol, 20:1548–1549","Mathieu, B., Cêtre-Sossah C., Garros C., Chavernac D., Balenghien T., Vignes-Lebbe R., Ung V., Candolfi E., Delécolle J. C. (2010). IIKC: An Interactive Identification Key for female Culicoides (Diptera: Ceratopogonidae) from the West Palearctic region. In Proceedings of the international congress Tools for Identifying Biodiversity: Progress and Problems: 20-22 September; Paris. Edited by Nimis PL, Vignes-Lebbe R; 201–205","Goffredo, M., & Meiswinkel R. (2004). Entomological surveillance of bluetongue in Italy: methods of capture, catch analysis and identification of Culicoides biting midges. Vet. Ital., 40 (3), 260–265","Borkent, A. (2017). Ceratopogonidae. In: Kirk-Spriggs AH, Sinclair BJ, editors. Manual of Afrotropical Diptera, Volume 2. Nematocerous Diptera and lower Brachycera Suricata 5. Pretoria: South African National Biodiversity Institute; p. 733–812","Purse, B. V., Carpenter S., Venter G. J., Bellis G., Mullens B. A. (2015). Bionomics of temperate and tropical Culicoides midges: knowledge gaps and consequences for transmission of Culicoides-borne viruses. Annu Rev Entomol, 60:373–92.","Carpenter, S., Mellor P. S., Fall A. G., Garros C., Venter G. J. (2017). African horse sickness virus: history, transmission, and current status. Annu Rev Entomol, 62:343–58.","Mellor, P. S., Boorman J., Baylis M. (2000). Culicoides biting midges: their role as arbovirus vec-tors. Annu Rev Entomol, 45:307–40.","Akakpo, A. J., Wombou Toukam C. M., Mankor A., Ly C. (2011). Economic impact of African horse sickness outbreak in Senegal in 2007. Bull Anim Hlth Prod Afr., 59:1–16.","Diouf, N. D., Etter E., Lo M. M., Lo M., Akakpo A. J. (2012). Outbreaks of African horsesickness in Senegal, and methods of control of the 2007 epidemic. Vet Rec., 172:152.","Kasap, O. E., Votýpka J., Alten B. (2013). The distribution of the Phlebotomus major complex (Dip-tera: Psychodidae) in Turkey. Acta Trop, 127:204–11","Folmer, O., Black M., Hoeh W., Lutz R., Vrijenhoek R. (1994). DNA primers for amplifcation of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 3:294–299.","Gunay, F., Alten B., Simsek F., Aldemir A., Linton Y. M. (2015). Barcoding Turkish Culex mos-quitoes to facilitate arbovirus vector incrimination studies reveals hidden diversity and new poten-tial vectors. Acta Trop, 143:112–20.","Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis pro-gram for Windows 95/98/NT. Nucleic Acids Symp Ser. 41:95–8.","Tamura, K., Stecher G., Peterson D., Filipski A., Kumar S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol., 30:2725–9.","Puillandre, N., Lambert A., Brouillet S., Achaz G. (2012). ABGD, automatic barcode gap discovery for primary species delimitation. Mol Ecol., 21:1864–77.","Librado, P., Rozas J. (2009). DnaSP v5: a software for comprehensive analysis of DNA polymor-phism data. Bioinformatics, 25:1451.","WHO. (1990). Control of the leishmaniasis. WHO TRS No. 793 report of a meeting of the WHO Expert Committee on the Control of Leishmaniasis, Geneva, 6–10 February 1989. Geneva: World Health Organization; 1990.","Dvorak, V., Kasap O. E., Ivovic V., Mikov O., Stefanovska J., Martinkovic F., Omeragic J., Pajovic I., Baymak D., Oguz G., Hlavackova K., Gresova M., Gunay F., Vaselek S., Ayhan N., Lestinova T., Cvetkovikj A., Soldo D. K., Katerinova I., Tchakarova S., Yılmaz A., Karaoglu B., Iranzo J. R., Kadriaj P., Velo E., Ozbel Y., Petric D., Volf P., Alten B. (2020). Sand flies (Diptera: Psychodidae) in eight Balkan countries: historical review and region-wide entomological survey. Parasit Vectors. 13(1):573. doi: 10.1186/s13071-020-04448-w.","Campbell, J. A., Pelham-Clinton E. C. (1960). A taxonomic review of the British species of Culi-coides Latreille (Diptera, Ceratopogonidae). Proc R Soc Edingb B. 1960;67:181–302","Becker, N., Petric D., Zgomba M., Boase C., Dahl C., Madon M., Kaiser A. (2010). Mosquito and Their Control, 2nd ed. Springer: Berlin/Heidelberg, Germany, 577 p.","Xiang, B., Kochar T. D. (1991). Comparison of mitochondrial DNA sequences of seven morphospe-cies of black flies Diptera. Genome, 34:306–311.","Wells, J. D., Stevens J. R. (2008). Application of DNA based methods in forensic entomology. An-nual Review of Entomology, 53:103–20","Sperling, F. A. H., Anderson G. S., Hickey D. A. (1994). A DNA-based approach to the identifica-tion of insect species used for postmortem interval estimation. Journal of Forensic Science. 39(2):418–427.","Kambhampati, S. (1995). A phylogeny of cockroaches and related insects based on DNA sequence of mitochindrial ribosomal RNA genes. Proceedings of National Academy Science, 92:2017–2020.","Tang, J., Pruess K., Cupp E. W., Unnasch T. R. (1996). Molecular phylogeny and typing of blackflies Diptera: Simuliidae that serve as vectors of human or bovine onchocerciasis. Medical Veterinary and Entomology, 10:228-234","Campbell, J., & Pelham-Clinton, E. (1960). X.—A Taxonomic Review of the British Species of Cu-licoides Latreille (Diptera, Ceratopogonidæ). Proceedings of the Royal Society of Edinburgh. Sec-tion B. Biology, 67(3), 181–302","Hernández-Triana, L. M., Crainey J. L., Hall A., Fatih F., Mackenzie-Dodds J., Shelley A. J., Zhou X., Post R. J., Gregory R. T., Hebert D. N. (2012). The utility of DNA barcoding for species identi-fication within the blackfly Subgenus Trichodagmia Enderlein (Diptera: Simuliidae: Simulium) and related taxa in the New World. Zootaxa 3154: 43–69.","Hernández-Triana, L. M., Brugman V. A., Prosser S. W. J., Weland C., Nikilova N., Thorne L., de Marco M. F., Fooks A. R., Johnson N. (2017). Molecular approaches for blood meal analysis and species identification of mosquitoes (Insecta: Diptera: Culicidae) in rural locations in southern England, United Kingdom. Zootaxa 4250: 067–076.","Hernández-Triana, L. M., Prosser S. W., Rodríguez-Pérez M. A., Chaverri L. G., Hebert P. D. N., Gregory T. R. (2014). Recovery of DNA barcodes from blackfly museum specimens (Diptera: Sim-uliidae) using primer sets that target a variety of sequence lengths. Molecular Ecology Resources 4: 508–18."]}

This study examined the activities of aspartate aminotransferase (ASAT), alanine aminotrans-ferase (ALAT), alkaline phosphatase (ALP), acid phosphatase (ACP), albumin (Alb), total protein (TP), creatin-phosphokinase (CPK), total lipid (TL), calcium (Ca) in the plasma of 304 dairy Holstein breed cows and their calves. The cows were held in an intensive farm breeding premises. Their meal consisted of ordinary alfalfa, silage, hay and concentrates with energy and protein supplements. The Student t-test showed significant difference (P < 0.05) in mean of the ASAT, ALAT, ALP, ACP, Alb, TP, TL, Ca between cows and calves of the breed. Mean values of the ASAT, ALAT, Alb, TP, TL and Ca was higher in cow group than in calves group (P < 0.05), while ALP and ACP was lower in cow in comparison to the calves group (P < 0.05).

Introduction The study of histopathological changes caused by influenza A (H5N8) viral infection in bird species is essential for the understanding of their role in the spread of this highly infectious virus. However, there are few such studies under natural conditions in minor gallinaceous species. This article describes the pathomorphological findings in Colchis pheasants infected naturally with H5N8 during an epizootic outbreak in Bulgaria. Material and Methods Samples of internal organs of 10 carcasses were collected for histopathological and immunohistochemical evaluation, virus isolation and identification, and nucleic acid detection. Results Consistent macroscopic findings were lesions affecting the intestine, heart, lung, and pancreas. Congestion and mononuclear infiltrate were common findings in the small intestine, as were necrosis and lymphoid clusters in the lamina propria of the caeca. Congestion with small focal necrosis and gliosis with multifocal nonpurulent encephalitis were observed in the brain. Myocardial interstitial oedema and degenerative necrobiotic processes were also detected. Immunohistological analysis confirmed systemic infection and revealed influenza virus nucleoprotein in all analysed organs. Conclusion Variable necrosis was observed in the brain, liver, trachea, heart, small intestine, and caeca. Viral antigen was commonly found in the brain, heart, lung and trachea. Contact with migrating waterfowls was suspected as a reason for the outbreak.

This article outlines pathomorphologic findings of a study involving commercial mule ducks with confirmed influenza A H5N8 infections after a series of outbreaks in Bulgaria. Examinations were carried out after performing necropsy on dead birds from three different age groups (up to 15, 20 to 30, and 40+ days of age) fattened on different farms. Among birds of all ages, gross lesions were present as lesions affecting the heart. Histologically, the myocardium exhibited severe intermyofibrillar edema, moderate to massive hemorrhages, and degenerative changes. All lesions resulted in single or multiple and small to massive myocardial infarctions. Other affected organs included the brain, lungs, liver, spleen, and pancreas. Nonpurulent lymphocytic encephalitis was found postmortem in ducks that had shown prior clinical nervous signs. Among ducks of all ages, a viral antigen in the cardiomyocytes and the epithelium of air capillaries was found through immunohistochemical detection methods. The results of the present study allowed us to conclude that the highly pathogenic avian influenza A H5N8 viral infection may manifest itself as a systemic illness in commercial mule ducks with septicemic lesions, resulting in high morbidity and mortality rates of up to 100%. Pathomorphologic lesions were somewhat different from those previously reported in wild waterfowl.Evaluación patológica de brotes de infección por influenza A H5N8 en patos mula en Bulgaria. Este artículo describe los hallazgos patomorfológicos de un estudio que involucró patos mula comerciales con infecciones confirmadas de influenza A H5N8 después de una serie de brotes en Bulgaria. Los exámenes se llevaron a cabo después de realizar la necropsia en aves muertas de tres grupos de edad diferentes (hasta 15 días, de 20 a 30 días y más de 40 días de edad) engordadas en diferentes granjas. Entre las aves de todas las edades, las lesiones macroscópicas estaban presentes como lesiones afectando el corazón. Histológicamente, el miocardio exhibió edema intermyofibrillar severo, hemorragias moderadas a masivas y cambios degenerativos. Todas las lesiones resultaron en infartos de miocardio simples o múltiples y de pequeños a masivos. Otros órganos afectados incluyeron el cerebro, los pulmones, el hígado, el bazo y el páncreas. Se encontró encefalitis linfocítica no supurativa se encontró post mortem en patos que habían mostrado signos nerviosos clínicos previos. Entre los patos de todas las edades, se encontró un antígeno viral en los cardiomiocitos y el epitelio de los capilares aéreos a través de métodos de detección inmunohistoquímica. Los resultados del presente estudio nos permitieron concluir que la infección viral altamente patógena de la influenza aviar H5N8 puede manifestarse como una enfermedad sistémica en patos mulos comerciales con lesiones septicémicas, lo que resulta en altas tasas de morbilidad y mortalidad de hasta el 100%. Las lesiones patomorfológicas fueron algo diferentes de las reportadas previamente en aves acuáticas silvestres.

Avian embryos have been gaining an increasing scientific interest as a valuable model system for the experimental cancer research that could contribute to a significant reduction of the number of laboratory animals. In the present study, the liver lesions induced by N-nitrosodimethylamine and N-nitrosodiethylamine in 15I line, White Leghorn embryos were identified and studied by routine histopathological method s. Foci of altered hepatocytes with basophilic and eosinophilic phenotype, well known as preneoplastic alterations were identified in the avian embryonal livers after in ovo exposure to both N-nitroso compounds. These studies were further extended by histopathological, haematological and biochemical examinations on the effects of N-nitrosodimethylamine in chickens hatched from carcinogen-inoculated eggs. In addition to the preneoplastic lesions observed in the avian livers, proliferations of oval and hepatocellular carcinoma cells, with clearly expressed signs of malignancy were found. The in ovo application of the chemical carcinogen was found to affect both hematological and blood biochemistry parameters measured in experimental birds. The established conditions such as thrombocytopenia and increased levels of liver enzymes, as an essential part of the paraneoplastic syndrome, were associated with the process of hepatocarcinogenesis. The results of this study confirm the preneoplastic nature of the focal lesions in embryonal avian liver and their progression to liver neoplastic alterations after a single in ovo application of known hepatocarcinogens. Moreover, the results indicate that 15I line, White Leghorn embryos are anew, valuable in ovo model for studies on hepatocarcinogenicity of chemical compounds and underline the importance of research on the development of different avian models of carcinogenicity.

The aim of this study is to estimate the histopathological changes in visceral organs of naturally infected with the avian influenza virus (AIV) subtype A H5N1 dalmatian pelicans in Bulgaria. The identified gross lesions are: haemorrhagic small intestine, sparse content in gizzard and proventriculus, well defined hyperemia of the tracheal mucosa associated with petechiae, as well as meningeal and brain congestion. The infected birds exhibited the following histopathological changes: edema of the tracheal mucosa with loss of mucosal glands, mild to moderate congestion with focal necrosis and multifocal non suppurative encephalitis and gliosis, mononuclear infiltration in the cecum, and diffuse mononuclear infiltration in the submucosa of the small intestine. The virus was detected by virus isolation (VI) and RT-PCR from tissue samples (lung, trachea, small intestine, brain, proventriculus, cloaca) from the infected birds.