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Epidemiologic study of environmental sources in a Prototheca zopfii outbreak of bovine mastitis.

Bovine mastitis represents the main form of occurrence of protothecosis in animals. The detection of mastitis caused by Prototheca sp. indicates a serious problem which can affect an entire herd. The purpose of this study is to explain some aspects of the epidemiology of mastitis due to Prototheca zopfii with the evaluation of the presence of these microorganisms in samples collected from potential sources in the dairy herd. This study was performed during a Prototheca zopfii outbreak of clinical bovine mastitis in the State of São Paulo, Brazil. The following samples were aseptically collected for microbiological examination: milk (n = 211); rectal swabs (from 15 calves and 2 lactating cows); swabs from tea cup rubbers during milking (n = 2); water (n = 6). Prototheca zopfii was isolated from 77 (36.49%) of the 211 milk samples; 11 calves and 2 cows showed Prototheca zopfii in faecal samples; both swabs collected from the tea cup rubbers showed viable forms of Prototheca zopfii; this microorganism was also isolated from 2 water samples, and 1 soil sample collected from the dry cow pasture. Prototheca zopfii seemed to be widespread throughout the dairy herd environment where this outbreak of bovine mastitis occurred.

Animals↗

Genomic and phenotypic characterization of mcr-9 carrying Enterobacter oligotrophicus recovered from bovine mastitis.

Bovine mastitis (BM) is a leading cause of economic loss in the dairy industry, driven by decreased milk yields, involuntary culling, and substantial veterinary costs. A single Gram-negative isolate recovered from BM was characterized in this study. For this, antimicrobial susceptibility testing (AST) was performed using the Neg-Urine-Combo 98 panel. Whole-genome sequencing (WGS) was employed to identify antimicrobial resistance genes (ARG), virulence factors (VF) genes, plasmid replicons and prophage sequences. Comparative genomic analysis was performed through phylogenetic analysis. The BM isolate was phenotypically identified as Citrobacter rodentium, however, WGS analysis reclassified the isolate as Enterobacter oligotrophicus. Phenotypic AST revealed a resistance profile of 12%, with the isolate exhibiting resistance to β-lactams antimicrobials, specifically amoxicillin-clavulanate, ampicillin and the cephalosporin-cefoxitin. Conversely, susceptibility was demonstrated for the remaining tested antimicrobials. Genomic profiling identified 31 ARG, 10 VF genes and 6 prophage sequences within the E. oligotrophicus genome. This study provides the first evidence of E. oligotrophicus as a causative agent of BM, expanding the known spectrum of pathogens affecting the dairy industry by delivering the second complete genome of E. oligotrophicus available globally. The identification of 31 ARG, 10 VF, and 6 prophage sequences underscore the potential pathogenic risk and environmental resilience of this isolate. These findings highlight the critical role of WGS-based surveillance in identifying non-conventional mastitis pathogens and underscore the need for targeted mitigation strategies in veterinary medicine.

Animals↗

Adherent and invasive Escherichia coli are associated with persistent bovine mastitis.

Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection. However, E. coli can also cause clonal persistent intramammary infection (IMI) in dairy cows. In this study, we explored the possibility that E. coli strains associated with persistent IMI are better able to adhere to, invade, survive and replicate in cultured mammary epithelial cells (MAC-T) than transient strains, and examined their serotype, overall genotype, phylogenetic group, and the presence of known virulence genes. Both transient and persistent E. coli strains adhered to MAC-T cells, but persistent strains invaded MAC-T cells 2.6-63.5 times more than transient strains. Blocking the adhesin/invasin FimH with mannose diminished but did not eliminate adhesion and invasion of any strain. Cytoskeletal and protein kinase inhibitors cytochalasin D, colchicine, genistein and wortmannin dramatically reduced invasion of MAC-T cells by both strains. All of the persistent strains, but only one transient strain, were able to survive and replicate intracellularly in MAC-T cells over 48 h. Transient and persistent strains displayed heterogeneous serotypes and overall genotypes, but similar phylogeny (group A), and lacked virulence genes of invasive E. coli. We have found that E. coli strains associated with persistent IMI are better able to invade and replicate within cultured mammary epithelial cells than transient strains. The invasion process involves the host cytoskeleton and signaling cascades and is not FimH dependent. Our findings suggest that the invasion of mammary epithelial cells and intracellular survival play an important role in the pathogenesis of persistent E. coli mastitis.

Analysis of Variance↗

Short communication: In vitro antimicrobial susceptibility of Prototheca wickerhamii and Prototheca zopfii isolated from bovine mastitis.

Bovine mastitis caused by Prototheca spp. can assume high significance because of economic losses and the potential risk to public health. Studies on the susceptibility of Prototheca spp. to antimicrobials have demonstrated its high level of resistance. We report the susceptibility of bovine isolates of Prototheca wickerhamii and Prototheca zopfii to amphotericin B and nystatin, 2 antifungal agents commonly used in the control of protothecosis, and discuss the results. After subculture, minimum inhibitory concentrations of both antifungal drugs were determined using macrodilution and agar diffusion methods. The inoculum concentration was standardized by determination of colony-forming units per milliliter. Nystatin showed more efficacy than amphotericin B in inhibiting P. wickerhamii growth. In contrast, growth inhibition of P. zopfii was similar for both antifungal agents. This study demonstrates different in vitro susceptibility patterns of P. wickerhamii and P. zopfii, reinforcing the necessity for more investigation into drugs that can be used with clinical efficacy.

Amphotericin B↗

Function and regulation of lymphocyte-mediated immune responses: relevance to bovine mastitis.

Bovine mastitis is one of the most costly diseases to the dairy industry. Prospects for effective vaccines are limited by the variety of microorganisms capable of causing mastitis. An understanding of the physiologic and immunologic factors controlling the susceptibility of the cow to disease will lead to more rational approaches to prevention and control. In this paper, we describe the basic components of the immune system, drawing upon information derived from studies with rodents and humans. Some of these findings have been confirmed in the bovine and other domestic species, and it is likely that further study will reveal additional similarities between the immune systems of laboratory animals, humans, and domestic animals. Some important differences have already been identified, such as altered lymphocyte circulation patterns in ruminant versus non-ruminant species. These differences are discussed. We describe the structural and functional properties of major histocompatibility complex antigens and their role in regulation of immune responses. Finally, we discuss the consequences of antigen-induced activation of T-lymphocytes and the role of these cells in response to disease-causing microorganisms.

Animals↗

Characterization studies on mycoplasmas isolated from bovine mastitis and the bovine respiratory tract.

Mycoplasmas isolated from bovine mastitis in California were classified into five distinct species. These included Mycoplasma bovis, M. bovigenitalium, M. alkalescens, M. canadenfe, and an unidentified strain, ST-6. Strains frequently recovered from the nose of young calves proved to be M. arginini, M. bovirhinis was recovered from the respiratory tract but was not a common finding.

Animals↗

Development of a rapid and sensitive test for identification of major pathogens in bovine mastitis by PCR.

Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g., S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using S. aureus DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 x 10(2) CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 x 10(3) CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.

Animals↗

Potentiation of antibiotic therapy for bovine mastitis by recombinant bovine interleukin-2.

Adjunct therapy with recombinant bovine interleukin-2 and antibiotics for Staphylococcus aureus IMI was investigated in an attempt to improve the therapy of antibiotics alone. Treatment of established S. aureus IMI with Na-cephapirin or Cefa-Lak produced average cures of 32.0 and 41.8%, respectively. When Na-cephapirin treatment was combined with recombinant bovine interleukin-2 at either 3.3 or 10 mg, the therapeutic efficacy was improved by an average of 20 to 30%. When Cefa-Lak treatment was combined with recombinant bovine interleukin-2 at 10 mg, the therapeutic efficacy was improved on average by 20%. Recombinant bovine interleukin-2, formulated in the excipient of the commercial Cefa-Lak, also improved the therapeutic efficacy by 16% compared with Cefa-Lak alone. Recombinant bovine interleukin-2, formulated in Cefa-Lak, maintained biological activity at room temperature for at least 21 d. After intramammary infusion of recombinant bovine interleukin-2, no biologically active interleukin-2 was detected in milk 48 h (four milkings) after administration. These data suggest that cytokines may be used as adjunct therapy with existing mastitis antibiotics or formulations of existing commercial products to improve the therapeutic efficacy.

Animals↗

Specific agglutination of Streptococcus agalactiae from bovine mastitis by casein components of bovine milk.

Streptococcus agalactiae strains freshly isolated from bovine mastitis cases clumped within 15 min of addition of small amounts of bovine milk to a broth culture. This reaction was not observed with isolates from human infections or bovine strains that had been maintained in the laboratory for extensive periods. Intensity of the clumping response as measured by a microtiter dilution assay was highly variable. Analysis of several single colony isolates derived from one strain indicated that the clumping phenotype was genetically unstable. The clumping reaction took place in the presence of rifampicin or chloramphenicol. Milk components that caused aggregation were heat stable, relatively insensitive to proteases, and were larger than 10,000 daltons. Purified casein also induced clumping in these strains.

Agglutination↗

Streptococcus uberis: a permanent barrier to the control of bovine mastitis?

The prevalence of bovine mastitis has been reduced over the past 25 years due to the implementation of a five-point control plan aimed at reducing exposure, duration and transmission of intramammary infections by bacteria. This has markedly reduced the incidence of bovine mastitis caused by bacteria which show a contagious route of transmission, but has had little effect on the incidence of mastitis due to bacteria which infect the gland from an environmental reservoir. Streptococcus uberis is one such bacterium which is responsible for a significant proportion of clinical mastitis worldwide. The inadequacies of the current methods of mastitis control have led to the search for additional measures, particularly vaccines to prevent intramammary infection by this bacterium. Such an approach requires detailed knowledge of the pathogenesis of intramammary infection. Our understanding of this area has grown in recent years but a lack of information still hampers disease control. Both live vaccines and, recently, crude sub-unit vaccines have shown promise against bovine mastitis due to S. uberis. Vaccines against mastitis must, however, be able to control infection without the participation of a marked inflammatory response. This review provides an overview of the recent advances which have been made in our understanding of host-pathogen interactions which promote infection and disease and highlights areas for strategic research aimed at controlling this bacterial infection.

Animals↗

Evaluation of tRNA intergenic spacer length polymorphism analysis as a molecular method for species identification of streptococcal isolates from bovine mastitis.

Sixty-nine bovine mastitis streptococci belonging to the species Str. agalactiae (n = 13), Str. dysgalactiae (n = 16), Str. canis (n = 22), Str. uberis (n = 20) and Str. parauberis (n = 4) and six reference strains of the five streptococcal species were examined for their tRNA gene intergenic length polymorphism (tDNA-ILP) fingerprint pattern. Epidemiologically unrelated isolates from bovine mastitis cases were selected by macrorestriction analysis with pulsed-field gel electrophoresis (PFGE). Their results were compared with those obtained from biochemical and serological studies and with those obtained by PCR-mediated identification amplifying species-specific gene segments of the five streptococcal species. According to the present results tDNA-ILP allowed a correct identification of all Str. agalactiae, Str. uberis and Str. parauberis strains investigated also including the reference strains of each species showing species-specific banding pattern. However, all Str. dysgalactiae ssp. dysgalactiae and all Str. canis strains appeared with an undistinguishable pattern which did not allow an identification of the species.

Animals↗

Viral infections and bovine mastitis: a review.

This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or parainfluenza 3 virus-induced clinical mastitis, while an intramammary inoculation of foot-and-mouth disease virus resulted in necrosis of the mammary gland. Subclinical mastitis has been induced after a simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4. Bovine leukaemia virus has been detected in mammary tissue of cows with subclinical mastitis, but whether this virus was able to induce bovine mastitis has not been reported. Bovine herpesvirus 2, vaccinia, cowpox, pseudocowpox, vesicular stomatitis, foot-and-mouth disease viruses, and bovine papillomaviruses can play an indirect role in the aetiology of bovine mastitis. These viruses can induce teat lesions, for instance in the ductus papillaris, which result in a reduction of the natural defence mechanisms of the udder and indirectly in bovine mastitis due to bacterial pathogens. Bovine herpesvirus 1, bovine viral diarrhoea virus, bovine immunodeficiency virus, and bovine leukaemia virus infections may play an indirect role in bovine mastitis, due to their immunosuppressive properties. But, more research is warranted to underline their indirect role in bovine mastitis. We conclude that viral infections can play a direct or indirect role in the aetiology of bovine mastitis; therefore, their importance in the aetiology of bovine mastitis and their economical impact needs further attention.

Animals↗

Cost benefit analysis of bovine mastitis in the UK.

Bovine mastitis reduces the yield and quality of milk and increases the rate of culling and veterinary costs. This reduces the profitability of farm milk production but the calculation of the extent of this economic loss is complex because of the many factors involved and deficiencies in the evidence on the relationship between the disease and various production factors. This paper examines the available evidence for the UK and provides a consistent analytical framework within which the benefits arising from reduced mastitis in dairy herds constrained by quota can be considered. It is estimated that since 1970 the farms that have followed the recommended control procedures have reduced the average annual number of cases of clinical mastitis from 135 to 40 cases/100 cows each year, while the quarters remaining uninfected for a whole year has increased from 65 to 80% of the total quarters. The costs of the main control procedures (e.g. 8.60 pounds/cow for dry-cow therapy and teat dipping or spraying) are broadly covered by the reduction in clinical mastitis, leaving the benefits of reduced subclinical infection (e.g. 3810 pounds for a 100 cow herd unconstrained by quota and achieving the average reduction in infection) as a substantial bonus. The imposition of quotas reduces the financial benefit of mastitis control but it still remains a worthwhile investment. The results of this analysis can be used to suggest maximum costs of additional new control measures produced by research. It also indicates that there is considerable value in production research which gives more precise knowledge of production systems, thus allowing producers to respond optimally to quota cuts.

Animals↗