ABSTRACT Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae are increasingly reported worldwide, emphasizing the need for rapid diagnostic tools to limit their spread and prevent outbreaks. Although CTX-M enzymes remain predominant, less common ESBLs such as GES, PER, and VEB are emerging and gaining clinical relevance. We developed simplex and multiplex lateral flow immunoassays (LFIAs) for the rapid detection of these ESBLs. Monoclonal antibodies were produced and incorporated into dedicated LFIA strips. The assays were evaluated using 116 WGS-characterized gram-negative bacteria (GNB) isolates expressing diverse β-lactamases and grown on various media. For testing, three colonies were suspended in 150 µL of extraction buffer, applied to the strips, and results were visually interpreted after 15 min. Both simplex and multiplex LFIAs achieved 100% sensitivity and specificity for detecting GES-, PER-, and VEB-producing isolates, irrespective of bacterial species, β-lactamase content, or growth conditions. The panel included prevalent variants of PER (n = 5, including PER-2), VEB (n = 9), and GES (n = 9, including ESBLs and carbapenemases). Detection limits for recombinant PER-1, VEB-1, and GES-1 were 5, 50, and 100 pg/mL, respectively, and 105, 106, and 106 CFU/mL for corresponding Acinetobacter baumannii producers grown in tryptic soy broth. Overall, these simplex and multiplex GES-, PER-, and VEB-targeting LFIAs provide a rapid, accurate, and user-friendly approach suitable for routine use in clinical microbiology laboratories. When combined with the NG-Test CTX-M MULTI assay, which detects the five major CTX-M groups, they may further enhance the overall detection of the most common ESBLs, particularly among non-fermenting GNB. IMPORTANCE Although less prevalent globally than CTX-M-type ESBLs, GES-, PER-, and VEB-type β-lactamases represent an important and often underrecognized mechanism of resistance, particularly in non-fermenting gram-negative pathogens. These enzymes are frequently associated with multidrug-resistant organisms and may significantly compromise available therapeutic options. The detection of the genes encoding these enzymes is often based on homemade PCR. Rapid diagnostic tests based on lateral flow immunoassays (LFIAs) have been successfully used to detect CTX-M-like ESBLs and the five main carbapenemases. Here, we demonstrated that GES, PER, and VEB enzymes can be readily detected by simplex or multiplex LFIAs from Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii colonies. These assays are rapid (≤15 min), highly sensitive (105–106 CFU/mL), requiring no specialized equipment, thus offering a user-friendly alternative to molecular assays in clinical laboratories. These features align well with the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, and Deliverable). Although less prevalent globally than CTX-M-type ESBLs, GES-, PER-, and VEB-type β-lactamases represent an important and often underrecognized mechanism of resistance, particularly in non-fermenting gram-negative pathogens. These enzymes are frequently associated with multidrug-resistant organisms and may significantly compromise available therapeutic options. The detection of the genes encoding these enzymes is often based on homemade PCR. Rapid diagnostic tests based on lateral flow immunoassays (LFIAs) have been successfully used to detect CTX-M-like ESBLs and the five main carbapenemases. Here, we demonstrated that GES, PER, and VEB enzymes can be readily detected by simplex or multiplex LFIAs from Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii colonies. These assays are rapid (≤15 min), highly sensitive (105–106 CFU/mL), requiring no specialized equipment, thus offering a user-friendly alternative to molecular assays in clinical laboratories. These features align well with the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, and Deliverable).

Simple Summary Feline patients may frequently develop anemia in association with a variety of underlying concurrent conditions, primarily as a direct consequence of metabolic disorders or infectious agents such as hemoplasmas. Additionally, anemia may arise as a part of a primary myeloproliferative disease, particularly associated with retroviral infections. Recent data from owners and veterinarians indicate that feline hemoplasmosis, despite its worldwide occurrence, is not routinely considered as the differential diagnosis for anemia in cats of Bosnia and Herzegovina. The infection is often underdiagnosed and overlooked, primarily due to the absence of reliable rapid testing methods and lack of hemoplasmosis PCR testing availability in Bosnia and Herzegovina, prior to this study. This is the first reported case of PCR-confirmed hemoplasmosis in Bosnia and Herzegovina. It further provides detailed information on patients’ signalment, clinical and molecular diagnostic approaches, treatment, and long-term follow-up. Despite being immunocompromised due to feline immunodeficiency virus infection, no recurrence of hemoplasmosis or retroviral reactivation was observed over an almost five-year period. This case also emphasizes the importance of considering hemoplasmosis in anemic cats, particularly those that do not respond to the treatment, and demonstrates the value of molecular methods in establishing definitive diagnosis.

Leptospirosis, a reemerging zoonotic disease caused by pathogenic bacteria of the genus Leptospira, affects a wide range of domestic and wild animals. The only investigation into sheep leptospirosis in Bosnia and Herzegovina was conducted nearly 50 years ago. This study aimed to assess the seroprevalence of leptospirosis and to identify the most common serovars in sheep in Bosnia and Herzegovina, using the microscopic agglutination test (MAT). Leptospirosis seroprevalence was determined to be 2.16% at a cut-off titer of ≥1:100 (55/2542) and 8.10% at a cut-off of ≥1:25 (206/2542), with all positive cases related to a single serovar. The MAT titers were 1:25 and 1:100, with the majority of positive animals having low titer (1:25) (151/206; 73.3%). At a cut-off of ≥1:100, the sera most frequently reacted to Pomona (54.55%) and Hardjo (27.27%), and less commonly to Saxkoebing and Icterohaemorrhagiae (0.2%) (P<0.05). Odds of seropositivity were higher for Pomona and Hardjo than for Saxkoebing and Icterohaemorrhagiae. The results of this study showed for the first time in Bosnia and Herzegovina, the presence of serovar Icterohaemorrhagiae in sheep, with Pomona and Hardjo as the dominant serovars. Although the seroprevalence is low, the potential zoonotic risk requires continuous monitoring and control strategies to prevent the spread of leptospirosis.

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.

Simple Summary Mastitis is defined as the inflammation of the mammary gland and is one of the most widespread and economically important diseases of dairy cows. Bacteria are the most reported mastitis-causative agents, while other pathogens are often overlooked because they are not routinely investigated. Incomplete diagnosis may result in inappropriate antimicrobial therapy, treatment failure, antimicrobial resistance, dissemination of pathogens, and mastitis recurrences. Thus, this study aimed to investigate the presence of not only bacteria but also other microorganisms associated with cattle mastitis on dairy farms in Bosnia and Herzegovina, a country that lacks an effective mastitis control programme and bacteriological analysis of mastitic milk. The current study revealed Mycoplasma bovis as the main pathogen and a variety of other mastitis-causing agents in cattle: bacteria (Escherichia coli, Staphylococcus aureus, coagulase negative staphylococci, Streptococcus agalactiae, Streptococcus uberis, and others), fungi (Candida spp.), and algae (Prototheca zopfii). The finding of mastitis cases requiring currently unavailable treatment and vaccines emerges in the broader scope of etiological agents in routine mastitis diagnosis. These measures applied at the herd and national levels are crucial for more effective mastitis control, animal health and welfare, the dairy industry, and public health. Abstract 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.

Little is known about vaginal and uterine bacteria in clinically healthy cats and their correlation with different stages of the oestrus cycle. The differences in vaginal bacterial flora between household and stray queens remains unknown. The aim of this study was to investigate the occurrence of vaginal and uterine bacteria in clinically healthy household and stray queens and to correlate culture findings with specific stages of the oestrous cycle. Vaginal and uterine samples from 40 clinically healthy queens were collected for isolation of bacteria and cytological examination. Bacteria were isolated from 31 vaginal swabs (77.5%) from stray (16/20; 80%) and household (15/20; 75%) cats. The isolates were more frequently detected in pure culture (18/31; 58%) than in mixed cultures (13/31; 41.9%). Streptococcus spp. was the most commonly identified bacteria (n = 16; 51.6%), followed by coagulase negative Staphylococcus spp. (n = 15; 48.4%) and E. coli (n = 12; 38.7%). A mixed bacterial culture of E. coli and Streptococcus spp. was commonly detected (50%), mainly in households (66.7%), whereas a mixed culture of Staphylococcus spp. and Streptococcus spp. (41.6%) was commonly isolated from stray cats (60%).  The frequency of isolation of pure or mixed bacterial cultures and the isolates did not vary significantly during the different stages of the oestrus cycle. All uterine samples tested were negative for bacteria. This study identified the most common bacteria in the vagina of clinically healthy cats. The isolation of vaginal bacteria in pure or mixed cultures should be considered as normal finding. The stage of the oestrous cycle apparently does not affect vaginal bacterial flora. Vaginal bacteria may differ between stray and households cats. No bacteria can be isolated from the uterus of clinically healthy cats.

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. ABSTRACT 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.

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.

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.

Little is known about the presence of methicillin-resistant Staphylococcus aureus (MRSA) in a canary bird (Serinus canarius domesticus). Although MRSA in a canary bird was previously reported, to the best of our knowledge, this is the first full description of the isolation and antibiotic resistance pattern of MRSA in this pet bird. A swab was taken from the nonhealing wound on the lateral thigh of a four-year-old, caged, housed alone, male canary bird. After the identification of Staphylococcus aureus, the antibiotic susceptibility profile of the isolate was obtained by the disk diffusion test. According to the resistance to Oxacillin and Cefoxitin, the isolate was identified as MRSA. The mecA gene was confirmed by PCR. The bird was treated by offering drinking water medicated with an injectable enrofloxacin formulation at 200 mg/L over 10 days period. Two weeks after therapy, intensive contraction of the wound was observed with a reduction in size. A week later, the complete epithelization of the wound defect was verified. In this study, we could not confirm the source of infection in a canary bird, but we believe that transmission was from wild birds when the cage was putting out in order to allow the bird to sunbathe or more likely via contact with the owner. The results of this study underline the necessity for further investigations on the epidemiological role of canary birds as potential reservoirs of MRSA.