Seroprevalence of leptospirosis among stray dogs in Bosnia and Herzegovina.

Leptospirosis is a (re) emerging zoonosis that occurs worldwide. This study aimed to assess seroprevalence of leptospirosis and to identify the most common reactive serovars and risk factors for seropositivity in apparently healthy stray dogs of unknown vaccination status in the Sarajevo region of Bosnia and Herzegovina. Positive microscopic agglutination test titres (≥ 1:25) were detected in 3.87% (156/4028) of samples and most of the sera reacted against one serovar (85.9%). Dogs were most commonly reactive to Canicola (40.4%) and Hardjo (33.3%), followed by Pomona (15.4%) Tarassovi (14.7%), Icterohaemorrhagiae (8.3%), Grippotyphosa (5.8%), Bratislava (1.3%) and Saxkoebing (0.6%). Dogs older than one year had higher odds of seropositivity compared to younger dogs. The seropositivity was higher in spring and autumn than in summer. These results advocate for the need of a control strategy for this zoonosis in the country, which should include sero-surveillance, monitoring, and the inclusion of additional serovars in the testing.

Pathogens Associated with Bovine Mastitis: The Experience of Bosnia and Herzegovina.

To obtain improved insights into the complex microbial aetiology of bovine mastitis, this study investigated the pathogens involved in cattle mastitis in Bosnia and Herzegovina. A total of 179 milk samples from cows with clinical mastitis (CM) and subclinical mastitis (SCM), as well as eight bulk tank milk (BTM) samples from 48 dairy farms, were analysed by standard bacteriological and mycological methods. Mycoplasma detection and identification were performed using culture techniques and real-time polymerase chain reaction (PCR). A total of 88 (49.2%) mastitis samples were positive for known mastitis pathogens at 32 of 47 farms (68.1%). Mycoplasma bovis was a predominant pathogen (25/187; 13.4%) in the majority of herds (14/48; 29.2%) and accounted for 48.9% of positive CM samples. Escherichia coli was the second most dominant CM pathogen (34%), followed by Streptococcus agalactiae (10.6%), whereas Staphylococcus aureus and coagulase-negative staphylococci were the most common in SCM samples (17.1%). Other mastitis pathogens included Candida spp. and Prototheca zopfii. Two BTM samples were positive for M. bovis only, and one was positive for a mixed culture of S. aureus and Streptococcus uberis. The finding of various causative agents of bovine mastitis, with M. bovis emerging as the main pathogen, emphasizes the significance of comprehensive testing that includes not only common mastitis pathogens but also mycoplasmas, fungi, and algae.

Complete Genome Sequences of Mycoplasma ovipneumoniae Strains 150 and 274, Isolated from Different Regions in Bosnia and Herzegovina.

Mycoplasma ovipneumoniae is an important pathogen in sheep, goats, and wild ruminants. We sequenced M. ovipneumoniae strains 150 and 274 from Bosnia and Herzegovina. Strain 150 has a circular genome of 1,053,380 bp with 29.15% GC content while strain 274 has 1,081,404 bp with 28.82% GC content.

Antimicrobial Resistance in Coagulase-Positive Staphylococci Isolated from Various Animals in Bosnia and Herzegovina.

The objective of this study was to determine and compare the antimicrobial resistance profiles of clinical Staphylococcus pseudintermedius (n = 90) and Staphylococcus aureus (n = 61) isolates from different animal species in Bosnia and Herzegovina. Among S. pseudintermedius isolates from dogs (n = 86), resistance to penicillin (87.2%) was most common followed by amoxicillin (76.7%) and amoxicillin/clavulanic acid (67.4%). A total of 25 isolates were found to be resistant to oxacillin of which 21 were mecA-positive and multidrug resistant (MDR). The most frequent MDR pattern was penicillins-lincosamides-cephalosporins-macrolides-fluoroquinolones. Among the 61 methicillin-susceptible S. pseudintermedius isolates, 18 were MDR (penicillins-lincosamides-macrolides). Most of S. aureus isolates were resistant to penicillin (50.8%), followed by amoxicillin (41%) and amoxicillin/clavulanic acid (31%). Resistance to cefoxitin was detected in only two isolates. All S. aureus isolates were mecA and mecC-negative. MDR was observed in six S. aureus isolates of which five were from cattle (penicillins-lincosamides-fluoroquinolones). Isolates from red foxes (Vulpes vulpes) were sensitive to most of the antimicrobials tested. The high number of methicillin-resistant S. pseudintermedius and MDR isolates in dogs exposed in this study underlines the urgent need for establishment of national antimicrobial resistance surveillance program in animals in the country, as well as for the surveillance of veterinary antimicrobial consumption.

Seroprevalence of Anaplasma spp. and Ehrlichia spp. and molecular detection of Anaplasma phagocytophilum and Anaplasma platys in stray dogs in Bosnia and Herzegovina.

Stray dogs may be highly exposed to vector-borne pathogens (VBPs), including zoonotic agents, and therefore may pose a high risk of spreading infections to other animals and humans. Among the Anaplasmataceae, Anaplasma phagocytophilum, A. platys and Ehrlichia canis are commonly identified species in dogs in Europe; however, information on the occurrence of these pathogens in canine populations from Bosnia and Herzegovina (B&H) is still lacking. Thus, the aim of this study was to determine the seroprevalence of Anaplasma spp. and Ehrlichia spp. in stray dogs in the Sarajevo region of B&H and to identify A. phagocytophilum, A. platys, E. canis and E. ewingii by molecular techniques. A total of 903 blood samples of stray dogs were screened by SNAP 4Dx Plus Test for the presence of antibodies against A. phagocytophilum/A. platys and E. canis/E. ewingii. Real-time PCR assays were performed for the detection of Anaplasmataceae, A. phagocytophilum, A. platys, E. canis and E. ewingii in seropositive dogs. Antibodies to A. phagocytophilum/A. platys and/or E. canis/E. ewingii were detected in 187 (20.7%) samples. Seroprevalence was highest for A. phagocytophilum/A. platys (184/903, 20.4%). Two dogs had antibodies to E. canis/E. ewingii, while one dog was found to have antibodies to A. phagocytophilum/A. platys and to E. canis/E. ewingii. Forty-eight (25.7%) of the 187 seropositive dogs examined by Real-time PCR were positive for Anaplasmataceae. A. phagocytophilum was detected in 45 (24%) samples, while one sample was positive for A. phagocytophilum and A. platys. Two samples positive for Anaplasmataceae tested negative in the species-specific PCRs. E. canis or E. ewingii could not be detected in any of the Ehrlichia-seropositive dogs. These findings highlight the need for dog health monitoring, improving the health and welfare of stray dog population, and establishment of effective surveillance systems to combat VBDs.

Molecular detection of Brucella spp. in clinical samples of seropositive ruminants in Bosnia and Herzegovina.

Brucellosis is one of the most common zoonotic diseases worldwide that is endemic to Bosnia and Herzegovina (B&H) and remains an emerging public and animal health concern in this country. Diagnostic testing of brucellosis in ruminants in B&H relies exclusively on serological methods. The present study was conducted to determine the presence of Brucella spp. in clinical samples of seropositive ruminants by Real-time PCR assay. Of the 135 samples tested, 37% were positive by Real-time PCR. The frequency of detection of Brucella-DNA in the samples collected from aborted animals (n = 20/20; 100%) was significantly higher (P < 0.00001) when compared to asymptomatic animals (n = 30/115; 26%). Among asymptomatic animals, 31.1% of cattle were positive for Brucella-DNA, followed by sheep (23.4%) and goats (16.6%). The results of this research underline the limitations of the current control policy and indicate the need for additional diagnostic methods required for more effective brucellosis control program.

Mycoplasma ovipneumoniae: A Most Variable Pathogen.

Mycoplasma ovipneumoniae, a well-established respiratory pathogen of sheep and goats, has gained increased importance recently because of its detection in wild ruminants including members of the Cervidae family. Despite its frequent isolation from apparently healthy animals, it is responsible for outbreaks of severe respiratory disease which are often linked to infections with multiple heterologous strains. Furthermore, M. ovipneumoniae is characterized by an unusually wide host range, a high degree of phenotypic, biochemical, and genomic heterogeneity, and variable and limited growth in mycoplasma media. A number of mechanisms have been proposed for its pathogenicity, including the production of hydrogen peroxide, reactive oxygen species production, and toxins. It shows wide metabolic activity in vitro, being able to utilize substrates such as glucose, pyruvate, and isopropanol; these patterns can be used to differentiate strains. Treatment of infections in the field is complicated by large variations in the susceptibility of strains to antimicrobials, with many showing high minimum inhibitory concentrations. The lack of commercially available vaccines is probably due to the high cost of developing vaccines for diseases in small ruminants not presently seen as high priority. Multiple strains found in affected sheep and goats may also hamper the development of effective vaccines. This review summarizes the current knowledge and identifies gaps in research on M. ovipneumoniae, including its epidemiology in sheep and goats, pathology and clinical presentation, infection in wild ruminants, virulence factors, metabolism, comparative genomics, genotypic variability, phenotypic variability, evolutionary mechanisms, isolation and culture, detection and identification, antimicrobial susceptibility, variations in antimicrobial susceptibility profiles, vaccines, and control.

Antimicrobial Susceptibility of Caprine and Ovine Mycoplasma ovipneumoniae Isolates.

The objective of this study was to determine the minimum inhibitory concentrations (MICs) of nine antimicrobials (enrofloxacin, ciprofloxacin, norfloxacin, gentamicin, spectinomycin, oxytetracycline, tylosin, florfenicol, and tiamulin) against 24 Mycoplasma ovipneumoniae isolates obtained from sheep and goats and to compare the resulting antimicrobial profiles. Enrofloxacin and ciprofloxacin had the lowest MIC50 values (<0.03 µg/mL) and MIC90 values (0.25 µg/mL) for all tested isolates. The highest MIC50 value (2 µg/mL) was obtained for florfenicol, while oxytetracycline and tylosin exhibited the highest MIC90 values (16 µg/mL). The MIC values for all fluoroquinolones and oxytetracycline were significantly lower for sheep isolates. Sheep isolates were considerably more susceptible to norfloxacin and tylosin than were goat isolates. This study demonstrated differences in antimicrobial susceptibilities between sheep and goat isolates, revealing M. ovipneumoniae in goat isolates to be less susceptible. The results suggest a possible link between antimicrobial profiles of M. ovipneumoniae isolates and their host ruminant species.

Genital mycoplasmas of healthy bitches.

Little is known about the presence of mycoplasmas in the genital tracts of domestic and stray bitches or in the vaginas of ovariohysterectomized (OHE) bitches. Moreover, to our knowledge, there has been no research to investigate the presence of canine vaginal mycoplasmas during the different stages of the reproductive cycle. We investigated the occurrence of mycoplasmas in the vaginas of healthy domestic and stray intact bitches, to correlate their presence with specific stages of the reproductive cycle, and to compare them with those in OHE bitches. We also investigated the presence of uterine mycoplasmas. Mycoplasmas were isolated from 41 of 122 vaginal swabs (34%) from domestic (27%) and stray (39%) bitches. Mycoplasma canis was the most commonly identified species ( n = 26; 63%), and was detected in both intact (60%) and OHE (73%) bitches. Mycoplasma isolates from the vaginas of healthy bitches did not vary during the various stages of the estrous cycle. Mycoplasmas were not detected in uterine samples.

Quantification of mycoplasmas in broth medium with sybr green-I and flow cytometry.

Mycoplasmas are the smallest and simplest organisms known. They form a large group of bacteria that can infect humans, animals, and plants. Even though several techniques have been proposed to enumerate mycoplasmas in broth medium, the determination of mycoplasma growth still remains a difficult task. The potential of using flow cytometry (FC) for rapidly estimating several species of mycoplasmas, M. agalactiae (Ma), M. putrefaciens (Mp), M. capricolum subsp. capricolum (Mcc), M. bovis (Mb), M. capricolum subsp. capripneumoniae (Mccp) and M. hyopneumoniae (Mh) in broth medium was examined. The FC analysis was performed by staining the mycoplasma cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count (Colony Forming Units--CFU) or Colour Changing Units (CCU) methods, depending on the mycoplasma species. There was a good correlation between mycoplasma counts determined by FC (cells ml(-1)) and by traditional plate count (CFU) or CCU methods. A correlation of 0.841, 0.981, 0.960, 0.913, 0.954, and 0.844 was obtained for Ma, Mp, Mcc, Mb, Mccp and Mh, respectively. FC method allowed results in 20-30 min, while 24-72 h was necessary for plate count method and 15 days for CCU method. FC was found to be a very useful, practical and fast technique to count mycoplasmas. These findings suggest that FC can be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasmas in broth medium.