In Vitro Activity of Quaternary Ammonium in Prototheca Isolated from Clinical Bovine Mastitis Identified by Mass Spectrometry and PCR Sequencing of the cytb Gene Marker.

The in vitro algaecide activity of quaternary ammonium (QA) against Prototheca isolated from bovine clinical mastitis was investigated, in which the clinical severity was scored, milk samples were subjected to microbiological culture, and algal species were identified by molecular typing. A total of 4275 milk clinical samples of different cows from ten large dairy farms were used. Forty-four (1%) samples of cows from three dairy farms yielded growth of Prototheca, of which 88.6% (39/44) were identified as Prototheca bovis and 11.3% (5/44) as Prototheca sp. by MALDI-TOF MS, whereas 100% of the isolates were identified as P. bovis using PCR sequencing of the cytb gene. Among cows for which clinical severity scoring was available, 78.8% (26/33) and 21.2% (7/33) had mild and moderate infections, respectively, whereas no animal showed severe clinical signs. The algaecide activity of QA in Prototheca was observed in low concentrations among all isolates, in 20.4% (9/44) at 35 ppm, 36.4% (16/44) at 17 ppm, and 43.2% (19/44) at an 8 ppm, in addition to activity on three reference Prototheca strains. Overall, the study highlights the predominance of P. bovis as the causative agent of algal mastitis in bovines. Prototheca induced abnormalities preponderantly in the milk and mammary gland tissue of cows, and to our knowledge, our study is the first to apply clinical severity scoring in protothecal mastitis. In addition, the study underlines the activity of QA in low concentrations against Prototheca, indicating its potential use as an antiseptic/disinfectant in milking facilities and dairy environments.

Flies (Insecta, Diptera) collected in the environment of dairy farms as carriers of Rotavirus A and betacoronavirus.

AIMS: We aimed to investigate the prevalence of rotavirus and coronavirus in dipterans that commonly inhabit the environment of dairy farms. METHODS AND RESULTS: We collected 217 insect specimens from nine dairy farms, which were examined through hemi-nested RT-PCR followed by Sanger sequencing in search of VP1 and N genes for rotavirus and bovine coronavirus-BCoV, respectively. With a predominance of Muscidae (152/217 = 70%) 11 families of Diptera were identified. Rotavirus A (RVA) and betacoronavirus (BCoV) were detected in 14.7% (32/217) and 4.6% (10/217) of the dipterans, respectively. Sequencing of the amplicons was possible for 11.5% (25/217) of RVA and 0.5% (1/217) of BCoV, confirming the presence of these pathogens. CONCLUSIONS: Our findings highlight the role of dipterans as carriers of RVA and BCoV of great relevance for public and animal health.

Genome-Based Characterization of Multidrug-Resistant Escherichia coli Isolated from Clinical Bovine Mastitis.

Mastitis occurrence in dairy cows is a broad topic that involves several sectors, from antimicrobial resistance and virulence of strains to economic implications and cattle management practices. Here, we assessed the molecular characterization (antimicrobial resistance determinants, virulence genes, sequences type, serotypes, and plasmid types) of 178 Escherichia coli strains isolated from milk samples from cows with clinical mastitis using a genome-based k-mers approach. Of these, 53 (29.8%) showed multidrug resistance by disc diffusion. We selected eight multidrug-resistant mastitis-associated E. coli for whole-genome sequencing and molecular characterization based on raw data using k-mers. We assessed antimicrobial resistance genes, virulence factors, serotypes, Multilocus Sequence Typing (MLST), and plasmid types. The most antimicrobial resistance gene found were blaTEM-1B (7/8), tetA (6/8), strA (6/8), strB (6/8), and qnrB19 (5/8). A total of 25 virulence factors were detected encoding adhesins, capsule, enzymes/proteins, increased serum survival, hemolysin, colicins, and iron uptake. These virulence factors were associated with Extraintestinal Pathogenic E. coli. Three pandemic clones were found: ST10, ST101, and ST69. Two E. coli were assigned in the O117 serogroup and one in the O8:H25 serotype. The most common plasmid groups were IncFII (7/8) and IncFIB (6/8). Our findings contribute to the knowledge of virulence mechanisms, epidemiological aspects, and antimicrobial resistance determinants of E. coli strains obtained from clinical mammary infections of cows.

Klebsiella-induced infections in domestic species: a case-series study in 697 animals (1997-2019).

Klebsiella species, particularly K. pneumoniae, are well-known opportunistic enterobacteria related to complexity of clinical infections in humans and animals, commonly refractory to conventional therapy. The domestic animals may represent a source of the pathogenic and multidrug-resistant Klebsiella species to humans. Nevertheless, most studies involving Klebsiella-induced infections in domestic animals are restricted to case reports or outbreaks. We retrospectively investigated selected epidemiological data, clinical aspects, and in vitro susceptibility pattern of 697 non-repetitive Klebsiella infections in livestock and companion species (1997-2019). The isolates were obtained from different clinical disorders from dogs (n = 393), cattle (n = 149), horses (n = 98), cats (n = 27), pigs (n = 22), sheep (n = 5), goats (n = 2), and buffalo (n = 1), except four isolates from subclinical bovine mastitis. Urinary (223/697 = 32%), enteric (117/697 = 16.8%), mammary (85/697 = 12.2%), reproductive (85/697 = 12.2%), and respiratory disorders (67/697 = 9.6%) were the most common clinical manifestations. Other miscellaneous clinical pictures (116/697 = 16.6%) included abscesses, otitis, hepatitis, conjunctivitis, pyodermitis, sepsis, and encephalitis. Norfloxacin (183/245 = 74.7%) and gentamicin (226/330 = 68.5%) were the most effective antimicrobials. High in vitro resistance of the isolates was seen to ampicillin (326/355 = 91.8%), amoxicillin/clavulanic acid (25/62 = 40.3%), and trimethoprim/sulfamethoxazole (100/252 = 39.7), and multidrug resistance to ≥ 3 classes of antimicrobials was found in 20.4% (142/697) isolates. Wide variety of clinical manifestations of Klebsiella-induced infections was observed, with a predominance of urinary, enteric, mammary, reproductive, and respiratory tract disorders, reinforcing opportunistic behavior of agent. Poor in vitro efficacy was observed to some conventional antimicrobials and ~ 20% of isolates exhibited resistance pattern, reinforcing the need for proper use of drugs on therapy approaches in domestic animals to avoid multidrug-resistant bacteria, an emergent global concern.

Accuracy of differences in blood and peritoneal glucose to differentiate between septic and non-septic peritonitis in horses.

Difference in blood and peritoneal glucose (DBPG) is used in clinical practice to support a diagnosis of septic peritonitis in horses. It is inexpensive, easy and rapid to perform. The aim of this study was to evaluate the accuracy of the DBPG to differentiate between septic and non-septic peritonitis in horses. Blood and peritoneal fluids were harvested from suspected animals. Plasma and peritoneal glucose levels, total nucleated cell count, direct microscopic and microbiological examinations of the peritoneal fluid were evaluated. Using DBPG levels, the animals were classified into two groups: difference ≥ 50 mg/dL (positive test) and difference < 50 mg/dL (negative test). Positive microbiological examination and/or presence of bacteria in direct microscopic examination was used as a gold standard to detect septic peritonitis. The accuracy parameters analysed were: sensitivity, specificity, and positive/negative predictive values, for which the results were respectively: 0.23, 0.91, 0.60 and 0.67. Due to poor accuracy, other cut-off margins and peritoneal glucose concentrations were evaluated. The test was considered most accurate when the DBPG was zero with sensitivity, specificity, and positive/negative predictive values of 0.85, 0.82, 0.73, 0.90 respectively. Peritoneal glucose concentrations alone were not a reliable feature to detect peritonitis. DBPG ≥50 mg/dL, widely used for the diagnosis of septic peritonitis, does not have a good accuracy and the DBPG = 0 has a better accuracy for detecting the disease.

Determinação da sensibilidade microbiana in vitro em linhagens de Enterococcus spp. isolados em leite de tanques de refrigeração / Determination of in vitro microbial sensitivity in Enterococcus spp. isolated from bulk tanks milk

Enterococos resist entes à vancomicina são frequentemente multirresistentes e tornaram-se uma das principais causas de infecções nosocomiais. Estão presentes causando mastites em rebanhos leiteiros e contaminando o leite de tanques de refrigeração. Porém, os dados são escassos em relação à sensibilidade das linhagens isoladas na produção leiteira. O objetivo do presente estudo foi determinar o perfil de resistência “in vitro” de Enterococcus spp. isolados de leite de tanques de refrigeração. Foram analisadas 194 amostras de 10 fazendas de Minas Gerais e São Paulo. Foi analisado o perfil de sensibilidade microbiana dessas linhagens frente a 14 antimicrobianos. Mais de 70% das amostras apresentaram resistência à cefalexina, cefoxitina, oxacilina e neomicina, 21,19% foram resistentes intermediárias a vancomicina.

Nontuberculous mycobacteria in milk from positive cows in the intradermal comparative cervical tuberculin test: implications for human tuberculosis infections.

Although the tuberculin test represents the main in vivo diagnostic method used in the control and eradication of bovine tuberculosis, few studies have focused on the identification of mycobacteria in the milk from cows positive to the tuberculin test. The aim of this study was to identify Mycobacterium species in milk samples from cows positive to the comparative intradermal test. Milk samples from 142 cows positive to the comparative intradermal test carried out in 4,766 animals were aseptically collected, cultivated on Lowenstein-Jensen and Stonebrink media and incubated for up to 90 days. Colonies compatible with mycobacteria were stained by Ziehl-Neelsen to detect acid-fast bacilli, while to confirm the Mycobacterium genus, conventional PCR was performed. Fourteen mycobacterial strains were isolated from 12 cows (8.4%). The hsp65 gene sequencing identified M. engbaekii (n=5), M. arupense (n=4), M. nonchromogenicum (n=3), and M. heraklionense (n=2) species belong to the Mycobacterium terrae complex. Despite the absence of M. tuberculosis complex species in the milk samples, identification of these mycobacteria highlights the risk of pathogen transmission from bovines to humans throughout milk or dairy products, since many of mycobacterial species described here have been reported in pulmonary and extrapulmonary diseases both in immunocompetent and immunocompromised people.

Diagnosis of mycobacteria in bovine milk: an overview.

Tuberculosis remains as the world's biggest threat. In 2014, human tuberculosis ranked as a major infectious disease by the first time, overcoming HIV death rates. Bovine tuberculosis is a chronic disease of global distribution that affects animals and can be transmitted to humans by the consumption of raw milk, representing a serious public health concern. Despite the efforts of different countries to control and eradicate bovine tuberculosis, the high negative economic impact on meat and milk production chains remains, given the decreased production efficiency (approximately 25%), the high number of condemned carcasses, and increased animal culling rates. This scenario has motivated the establishment of official programs based on regulations and diagnostic procedures. Although Mycobacterium tuberculosis and Mycobacterium bovis are the major pathogenic species to humans and bovines, respectively, nontuberculous mycobacteria within the Mycobacterium genus have become increasingly important in recent decades due to human infections, including the ones that occur in immunocompetent people. Diagnosis of mycobacteria can be performed by microbiological culture from tissue samples (lymph nodes, lungs) and secretions (sputum, milk). In general, these pathogens demand special nutrient requirements for isolation/growth, and the use of selective and rich culture media. Indeed, within these genera, mycobacteria are classified as either fast- or slow-growth microorganisms. Regarding the latter ones, incubation times can vary from 45 to 90 days. Although microbiological culture is still considered the gold standard method for diagnosis, molecular approaches have been increasingly used. We describe here an overview of the diagnosis of Mycobacterium species in bovine milk.

Successful therapy in unusual generalized Dermatophilus congolensis infection in a calf based on modified in vitro disk diffusion test / Sucesso no tratamento de infecção generalizada em bezerra por Dermatophilus congolensis baseado em teste in vitro modificado de difusão com discos

Bovine dermatophilosis is a dermatitis characterized by typical focal or localized lesions with "paintbrush" aspect and occasionally as disseminated cutaneous disease. We report the case of a one-year-old Nelore female with history of chronic cutaneous disseminated lesions that appeared immediately after a rainfall period. Serous to purulent exudates, hair with tufted appearance, hyperkeratotic, non-pruritic, hardened, yellowish to brown, and coalescent crusty lesions were observed distributed all over its body. Removal of the crusts revealed ulcerated or hemorrhagic areas, with irregular elevated crusts like "paintbrush". Microbiological diagnosis enabled the identification of a microorganism, the Dermatophilus congolensis. Despite disseminated and chronic lesions, we obtained a successful therapy with parenteral therapy using long-acting tetracycline based on modified in vitro disk diffusion test. The present report highlights success therapy in uncommon generalized bovine dermatophilosis with selection of first-choice drugs based on modified in vitro susceptibility test, and need of responsible use of antimicrobials in livestock.(AU)

A dermatofilose bovina é uma dermatite caracterizada por lesões focais ou localizadas com aspecto de "pincel" e, ocasionalmente, como lesão cutânea disseminada. Relata-se o caso de uma fêmea bovina de um ano de idade, que foi atendida apresentando história de lesões cutâneas crônicas imediatamente após um período de alta pluviosidade. Ao exame clínico, lesões serosas a purulentas, com hiperqueratose, coalescentes, não pruriginosas, ressecadas, de coloração amarelada à acinzentada foram observadas distribuídas de modo generalizado pelo animal. A remoção das crostas revelou áreas ulceradas ou hemorrágicas, com crostas irregulares e elevadas semelhantes a "pincel". O diagnóstico microbiológico possibilitou a identificação do micro-organismo Dermatophilus congolensis. Apesar das lesões disseminadas e crônicas, a cura do animal foi obtida com tratamento parenteral usando oxitetraciclina de longa duração, baseado em teste in vitro de sensibilidade microbiana modificado. O presente relato ressalta o sucesso no tratamento de caso incomum de lesões generalizadas de dermatofilose bovina com respaldo de teste in vitro de sensibilidade modificado, bem como a necessidade do uso responsável de antimicrobianos em animais de produção.(AU)

Diagnosis of mycobacteria in bovine milk: an overview

ABSTRACT Tuberculosis remains as the world’s biggest threat. In 2014, human tuberculosis ranked as a major infectious disease by the first time, overcoming HIV death rates. Bovine tuberculosis is a chronic disease of global distribution that affects animals and can be transmitted to humans by the consumption of raw milk, representing a serious public health concern. Despite the efforts of different countries to control and eradicate bovine tuberculosis, the high negative economic impact on meat and milk production chains remains, given the decreased production efficiency (approximately 25%), the high number of condemned carcasses, and increased animal culling rates. This scenario has motivated the establishment of official programs based on regulations and diagnostic procedures. Although Mycobacterium tuberculosis and Mycobacterium bovis are the major pathogenic species to humans and bovines, respectively, nontuberculous mycobacteria within the Mycobacterium genus have become increasingly important in recent decades due to human infections, including the ones that occur in immunocompetent people. Diagnosis of mycobacteria can be performed by microbiological culture from tissue samples (lymph nodes, lungs) and secretions (sputum, milk). In general, these pathogens demand special nutrient requirements for isolation/growth, and the use of selective and rich culture media. Indeed, within these genera, mycobacteria are classified as either fast- or slow-growth microorganisms. Regarding the latter ones, incubation times can vary from 45 to 90 days. Although microbiological culture is still considered the gold standard method for diagnosis, molecular approaches have been increasingly used. We describe here an overview of the diagnosis of Mycobacterium species in bovine milk.