Unpasteurized Cow's Milk Research Articles

Detection of Toxoplasma gondii DNA in milk of dairy cows from southern Brazil.

Consumption of unpasteurized cow's milk may be a transmission route for some pathogenic microorganisms, but there is little information about the risk of Toxoplasma gondii infection. Blood and milk samples were collected in a paired and random fashion from 106 dairy cows and bulk-tank milk samples were also collected from each of the six farms, in southern Brazil. Serum anti-T.gondii antibodies (IgG) were detected by an indirect fluorescent antibody test (IFAT) with a cutoff point of 1:64. Nested PCR targeting the ITS1 was performed on milk samples to detect the Sarcocystidae family, confirmed to be T.gondii by Sanger sequencing. The occurrence of anti-T.gondii antibodies in the herds was 14.1%, (15/106) with seropositive cows in all herds. Antibody titers in positive samples ranged from 64 to 128. T.gondii DNA was detected in 2.8% (03/106) of the milk samples. The ITS1 sequences generated in this study were ON809793 - ON809794 and the sequencing revealed 98-100% identity with T. gondii DNA sequences deposited in GenBank. All cows PCR positive for T.gondii in milk were negative for IgG antibodies in serum, suggesting that naturally infected cows may shed T. gondii in milk in the acute phase of infection. The results of this study demonstrate that T. gondii DNA may be detected in raw cow's milk, so the potential risks of lactogenic infection should be considered. The presence of T. gondii DNA in milk does not confirm that the protozoa are viable and infective, and further investigations into the role of cow's milk in the epidemiology of toxoplasmosis are needed.

DEVELOPMENT OF A MULTIANALYTE MASS SPECTROMETRY METHOD FOR QUANTITATION OF ANTIHYPERTENSIVES IN MILK

Objective: Hypertension in pregnancy is common and affects about 10% of women. One third of cases persist into the postnatal period requiring treatment with antihypertensive medications to control blood pressure. However, there is a lack of quality evidence to quantify the transfer of antihypertensives into human milk and to therefore recommend their safe use during breastfeeding. Current reviews on this subject are opinion driven or formed upon recommendations from expert working groups based on anecdotal case reports. No antihypertensives are licensed for use during breastfeeding in the UK and many have not been tested during pregnancy or breastfeeding. Design and method: A multi-marker liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS/MS) assay was developed and optimised to simultaneously quantify 24 antihypertensives and their metabolites (enalapril (and enalaprilat), lisinopril, perindopril, ramipril (and ramiprilat), trandolaprilat, azilsartan, candesartan, irbesartan, olmesartan, telmisartan, valsartan, atenolol, bisoprolol, carvedilol, labetalol, metoprolol, propranolol amlodipine, diltiazem, dehydronifedipine, eplerenone and bumetanide) in milk. Whole, unpasteurised cow's milk was used as surrogate matrix for method development. Analytes were extracted from milk by protein precipitation. Chromatographic analyses were performed on an Agilent Technologies Zorbax Rapid Resolution C18 column using gradient elution. Identification and quantitation were carried out using electrospray ionisation (positive) on an Agilent Technologies 6410 Triple Quad Mass Spectrometer by monitoring specific precursor and fragment ions in multiple reaction monitoring mode using internal standards. The “Standard Practices for Method Validation in Forensic Toxicology” criteria was used for validation. Results: The method passed validation with accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of < or = 15% at all tested concentrations for the analytes. The assay sensitivity of 10ng/ml was sufficient to detect the analytes in milk. Conclusions: To the best of our knowledge, this is the first assay that permits simultaneous quantitation of 24 commonly prescribed antihypertensives and their metabolites from milk to enable safe use in breastfeeding. The results could be used to guide research on which antihypertensives are safe to use whilst breastfeeding and which require an abstinence period prior to continuation of breastfeeding.

TB OR NOT TB

Introduction: Tuberculosis (TB) an infection caused by Mycobacterium tuberculosis and less frequently by ingestion of Mycobacterium bovis infected unpasteurized cow's milk or by other atypical mycobacteria1,is a very commonly encountered disease in the Indian subcontinent and must almost always be included in the differential diagnosis of an infective pathology since it can affect any organ most commonly lungs. Materials and methods: An 18 year old male presented to our tertiary care hospital with a right sided submandibular and sub-mental swelling. Differential diagnosis included abscess involving submandibular salivary gland/Ludwig's angina but with further evaluation, tuberculosis of submandibular lymph nodes was found to be the provisional diagnosis. Conclusion: Careful evaluation with detailed history, clinical examination and appropriate investigations are necessary before deciding the denitive management and tuberculosis, being highly prevalent in the Indian subcontinent, should always be considered as a possible diagnosis as it signicantly changes the course of treatment required by the patient

Evaluation of gamma-aminobutyric acid content in Portuguese cheeses with protected designation of origin status.

Health-conscious consumers are increasingly paying attention to healthy diets and focusing on natural bioactive compounds in foods and their effects on mental health. This opens new opportunities for the study of artisanal cheeses as biofunctional foods. In the work described in this Research Communication, the gamma-aminobutyric acid (GABA) content of seven different Portuguese cheeses produced from unpasteurized cow, sheep, and goat milk and granted with protected designation of origin (PDO) status was analysed. The PDO cheeses made from cow milk analysed in this study were São Jorge (3, 4, 7, 12 and 24 months of maturation) and Pico cheeses. PDO cheeses made from sheep milk were Serra da Estrela, Serpa, Nisa and Azeitão. Cheeses made from sheep and goat milk included Beira Baixa yellow cheese. The GABA content in the Azorean PDO cheeses (made from cow milk) ranged from 1.23 to 2.64 g/kg of cheese. Higher variations in GABA content were observed in cheeses made from sheep and goat milk (0.73-2.31 g/kg). This study provides information on the GABA content in different Portuguese PDO cheeses and shows that hard or semi-hard ripened cheeses are a suitable matrix for GABA production by lactic acid bacteria.

The Ability of Shiga Toxin-Producing Escherichia coli to Grow in Raw Cow's Milk Stored at Low Temperatures.

Despite the lack of scientific evidence, some consumers assert that raw milk is a natural food with nutritional and immunological properties superior to pasteurized milk. This has led to the increased popularity of unpasteurized cow milk (UPM) and disregard for the risks of being exposed to zoonotic infections. Dairy cattle are healthy carriers of Shiga toxin (Stx)-producing E. coli (STEC), and contaminated UPM has caused STEC outbreaks worldwide. The association between STEC, carrying the eae (E. coli attachment effacement) gene, and severe diseases is well-established. We have previously isolated four eae positive STEC isolates from two neighboring dairy farms in the Southeast of Norway. A whole genome analysis revealed that isolates from different farms exhibited nearly identical genetic profiles. To explore the risks associated with drinking UPM, we examined the ability of the isolates to produce Stx and their growth in UPM at different temperatures. All the isolates produced Stx and one of the isolates was able to propagate in UPM at 8 °C (p < 0.02). Altogether, these results highlight the risk for STEC infections associated with the consumption of UPM.

&lt;i&gt;Toxoplasma gondii&lt;/i&gt; Infection in Pregnant Women: Seroprevalence and Associated Risk Factors in Ouagadougou, Burkina Faso

Toxoplasma gondii (T. gondii) infection can lead to abortion in pregnant women. Unfortunately, very limited information is available concerning the seroprevalence and associated risk factors in pregnant women in Burkina Faso. Therefore, the present study aimed to estimate the seroprevalence of T. gondii among pregnant women attending for antenatal care. A cross-sectional study was conducted containing 579 pregnant in Ouagadougou, Burkina Faso. Serological patterns were assessed by enzyme-linked fluorescent immunoassay and the study utilized univariate analysis to identify the potential risk factors for T. gondii infection. Of the 579 pregnant women investigated, 29.71% were tested as T. gondii-seropositive, with 25.91% seropositive for T. gondii IgG antibodies, 1.9% positive for IgM and 1.9% positive for both IgM and IgG. We found firstly that pregnant women consuming unpasteurized cow's milk had a significantly higher seroprevalence than individuals who did not consuming (30.15% vs 06.67%; p=0.0039). Then, age was associated with an increased risk of being seropositive for T. gondii, seroprevalence increases significantly with age, ranging from 18% for 15-25 year olds to 45% for those over 35 years old, pT. gondii (p=0.0065 and 0.023, respectively). The other risk factors like contact with soil, drinking water source, contact with cats and eating some under-cooked meat were not significantly associated with T. gondii infection. Altogether, our study showed that seroprevalence of T. gondii was mainly related to consumption of unpasteurized cow's milk, to patient age, to the number of pregnancies and to living children. The findings will provide key and baseline data for prevention and control of toxoplasmosis among pregnant women and other people.

The maternal gut microbiome during pregnancy and offspring allergy and asthma

Environmental exposures during pregnancy that alter both the maternal gut microbiome and the infant's risk of allergic disease and asthma include a traditional farm environment and consumption of unpasteurized cow's milk, antibiotic use, dietary fiber, and psychosocial stress. Multiple mechanisms acting in concert may underpin these associations and prime the infant to acquire immune competence and homeostasis following exposure to the extrauterine environment. Cellular and metabolic products of the maternal gut microbiome can promote the expression of microbial pattern recognition receptors, as well as thymic and bone marrow hematopoiesis relevant to regulatory immunity. At birth, transmission of maternally derived bacteria likely leverages this in utero programming to accelerate postnatal transition from a TH2- to TH1- and TH17-dominant immune phenotype and maturation of regulatory immune mechanisms, which in turn reduce the child's risk of allergic disease and asthma. Although our understanding of these phenomena is rapidly evolving, the field is relatively nascent, and we are yet to translate existing knowledge into interventions that substantially reduce disease risk in humans. Here, we review evidence that the maternal gut microbiome impacts the offspring's risk of allergic disease and asthma, discuss challenges and future directions for the field, and propose the hypothesis that maternal carriage of Prevotella copri during pregnancy decreases the offspring's risk of allergic disease via production of succinate, which in turn promotes bone marrow myelopoiesis of dendritic cell precursors in the fetus.

Prevalence of allergic diseases and their symptoms in schoolchildren according to the birth mode.

The relationship between birth by cesarean section (CS) and allergic diseases' prevalence continues to be controversial. We aimed to investigate if being born by CS is associated with the prevalence of allergic diseases and their symptoms in schoolchildren. This study included children between 6 and 7 years of age, selected by population-based sampling. We investigated the presence of allergic diseases and their symptoms, family history of asthma, smoking in parents, breastfeeding, exposure to pets, the season of birth, number of siblings, consumption of unpasteurized cow's milk, and mode of birth. We included 1003 subjects (526 girls), of which 44.2% were born through CS. The prevalence of allergic diseases did not differ according to birth mode. Asthma in either parent, current smoking by the mother, breastfeeding, and unpasteurized cow's milk consumption were associated with wheezing at some time in life. No association between CS birth mode and allergic diseases and their symptoms was found.

Detection of methicillin-resistant coagulase-negative staphylococci and PVL/mecA genes in cefoxitin-susceptible Staphylococcus aureus (t044/ST80) from unpasteurized milk sold in stores in Djelfa, Algeria

This study was designed to determine antimicrobial resistance phenotypes and genotypes and virulence factors in Staphylococcus aureus and coagulase-negative staphylococci (CNS) in unpasteurized milk sold in Djelfa, Algeria. Eighty-two unpasteurized cow milk samples were randomly obtained from 82 retail stores in Djelfa and tested to detect staphylococci. Species were identified by biochemical tests and MALDI-TOF. Antimicrobial resistance phenotypes and genotypes were determined by disk diffusion test, PCR, and sequencing. The Staph. aureus isolates were subjected to spa typing, multilocus sequence typing, and detection of virulence genes and the scn gene by PCR and sequencing. Forty-five (54.9%) milk samples were contaminated by staphylococci and 45 isolates were recovered: 10 Staph. aureus (12.2% of total samples) and 35 CNS (42.7%). Resistance to penicillin (blaZ), tetracycline (tetL/tetK), and erythromycin (ermB/msrA/ermC) were the most common phenotypes (genotypes). Three CNS were methicillin-resistant and all were mecA-positive. The Staph. aureus isolates were ascribed to the following lineages [spa type/sequence type/associated clonal complex (number of isolates)]: t267/ST479/CC479 (n = 6), t1510/ST5651/CC45 (n = 1), t359/ST97/CC97/ (n = 1), t346/ST15/CC15 (n = 1), and t044/ST80 (n = 1). The mecA gene was detected in the cefoxitin-susceptible t044/ST80 isolate and co-harbored the lukF/lukS-PV and scn genes. The detection of mecA-PVL-positive Staph. aureus, methicillin-resistant CNS, and multidrug-resistant staphylococcal species indicates a potentially serious health issue and reveals that unpasteurized milk sold in Djelfa city could be a potential vehicle for pathogenic and antimicrobial-resistant staphylococci.

Risk factors for Toxoplasma gondii seropositivity in the Old Order Amish.

Toxoplasma gondii (T. gondii) is an important human disease-causing parasite. In the USA, T. gondii infects >10% of the population, accrues economic losses of US$3.6 billion/year, and ranks as the second leading culprit of foodborne illness-related fatalities. We assessed toxoplasmosis risk among the Old Order Amish, a mostly homogenous population with a high prevalence of T. gondii seropositivity, using a questionnaire focusing on food consumption/preparation behaviours and environmental risk factors. Analyses were conducted using multiple logistic regression. Consuming raw meat, rare meat, or unpasteurised cow or goat milk products was associated with increased odds of seropositivity (unadjusted Odds Ratios: 2.192, 1.613, and 1.718 , respectively). In separate models by sex, consuming raw meat, or consuming unpasteurised cow or goat milk products, was associated with increased odds of seropositivity among women; washing hands after touching meat with decreased odds of seropositivity among women (adjusted OR (AOR): 0.462); and cleaning cat litterbox with increased odds of seropositivity among men (AOR: 5.241). This is the first study to assess associations between behavioural and environmental risk factors and T. gondii seropositivity in a US population with high seroprevalence for T. gondii. Our study emphasises the importance of proper food safety behaviours to avoid the risk of infection.

Think before you drink: Fatal case of meningitis in an infant

Salmonella is a common cause of community-acquired gastrointestinal infection. Serious life-threatening infection such as meningitis is a rare occurrence. Both acute complications and long-term neurological sequelae can occur in infants with Salmonella meningitis, in whom mortality rates are high. Treatment with Ceftriaxone or Cefotaxime for 4 weeks or more is recommended for complete killing of the pathogen and to prevent relapse. We report a rare case of S. meningitis in a young infant, possibly acquired from the ingestion of unpasteurized cow milk.

Maternal farm exposure/ingestion of unpasteurized cow's milk and allergic disease: Erratum.

Maternal farm exposure/ingestion of unpasteurized cow's milk and allergic disease: Erratum.

Real-time quantitative PCR-based detection of Coxiella burnetii in unpasteurized cow's milk sold for human consumption.

Coxiella burnetii is a zoonotic pathogen with a worldwide distribution that is responsible for Q fever in humans. It is a highly infectious bacterium that can be transmitted from cattle to humans through the consumption of unpasteurized milk. We report the molecular identification of C.burnetii in raw cow's milk being sold directly for human consumption in Brazil without official inspection or pasteurization. One hundred and twelve samples of raw milk were analysed by real-time quantitative PCR (qPCR), and C.burnetii was detected in 3.57% (4/112) of the samples at a concentration ranging from 125 to 404 bacteria per millilitre. The identification of this zoonotic pathogen in raw milk sold directly for human consumption is a public health concern since C.burnetii can be transmitted through the oral route. This result indicates that health education and other preventive measures should be officially implemented in Brazil to prevent the spread of infection. To our knowledge, this is the first qPCR-based detection of C.burnetii in raw milk samples from cows sold in Brazil that do not undergo official inspection or pasteurization.

S. epidermidis strains from artisanal cheese made from unpasteurized milk in Poland - Genetic characterization of antimicrobial resistance and virulence determinants

In Poland artisanal cheese production is an important local economic activity. Artisanal cheese is usually produced using raw cow's milk, animal rennet and salt, without the addition of starter cultures. Coagulase negative staphylococci (CoNS) are often present in artisanal cheeses. Pathogenic potential of some CoNS species, especially S. epidermidis, suggests that they could correspond to emerging pathogens. The identified risk factors correspond to virulence, antibiotic resistance and biofilm formation. Therefore, we aimed to characterize S. epidermidis isolated along the artisanal raw milk production chain. Seventy artisanal cheeses samples from unpasteurized cow milk purchased in Podlasie and Warmia and Mazury region in Poland, were included in this study. A total of 26 S. epidermidis isolates were obtained. Most of them were antimicrobial resistant, such as to penicillin (84,6%), clindamycin (46,2%), tetracycline (42,3%), erythromycin (42,3%) and cefoxitin (26,9%). Only one isolate was susceptible to all antibiotics used in the study. All methicillin resistant S. epidermidis strains (26,9%) harbored mecA gene. Isolates, phenotypic resistant to tetracycline, harbored at least one tetracycline resistance determinant on which tet(M) was most frequent. Moreover, all tetracycline resistant strains harbored Tn916-Tn1545-like integrase family gene. In the erythromycin resistant isolates, the macrolide resistance genes ermC, ermB or msrA/B were present. Seven strains demonstrated a strong ability to form biofilm and moderate and weak biofilm was demonstrated by 4 strains, whereas 11 of S. epidermidis isolates were found to be unable to form a biofilm. All strains producing strong biofilm harbored the icaD gene which occurred independently or in combination with the icaA. Insertion element IS256, was identified in 15,4% of S. epidermidis strains, all of which were multidrug resistant. Arginine Catabolic Mobile Element (ACME) was identified in 13 of the 26 examined strains (50%). Most common was ACME type I (26,9%), followed by type III (15,4%) and type II (7,7%). Our data indicate that S. epidermidis are widely present in artisanal cheeses from raw whole cow milk in Poland. Many isolated strains containing more virulence factors and antibiotic resistant and carry mobile genetic elements which represent a potential source of resistance transmission to bacteria in humans.

Tick-borne encephalitis virus in cows and unpasteurized cow milk from Norway.

Tick-borne encephalitis virus (TBEV) is recognized as the most important zoonotic tick-transmitted virus in Europe. TBEV is mainly transmitted to humans through bites from TBEV-infected ticks (Ixodes ricinus and Ixodes persulcatus). However, alimentary infection after consumption of unpasteurized milk and cheese from domestic ruminants has been reported. There is little information about TBEV in ruminants in Norway. The objectives of this study were to analyse unpasteurized cow milk for TBEV RNA and to study the presence of IgG antibodies to TBEV in the same animals. A total of 112 milk and blood samples were collected from cows from five different farms spread from southern to northern Norway. The milk samples were analysed by an in-house reverse transcription (RT) real-time polymerase chain reaction and confirmed by pyrosequencing. Serum samples were screened by a commercial enzyme-linked immunosorbent assay and verified by a TBEV-specific serum neutralization test. We found TBEV RNA in unpasteurized milk collected from farms in the municipalities of Mandal, Skedsmo and Brønnøy in 5.4% of the tested animals. Specific antibodies to TBEV were only detected in Arendal, where 88.2% of the tested animals were positive. Further studies on milk containing TBEV RNA should be performed to conclude if TBEV found in unpasteurized milk in Norway is infectious, which could be of great importance in a One Health perspective.

Metagenomics of pasteurized and unpasteurized gouda cheese using targeted 16S rDNA sequencing

BackgroundThe microbiome of cheese is diverse, even within a variety. The metagenomics of cheese is dependent on a vast array of biotic and abiotic factors. Biotic factors include the population of microbiota and their resulting cellular metabolism. Abiotic factors, including the pH, water activity, fat, salt, and moisture content of the cheese matrix, as well as environmental conditions (temperature, humidity, and location of aging), influence the biotic factors. This study assessed the metagenomics of commercial Gouda cheese prepared using pasteurized or unpasteurized cow milk or pasteurized goat milk via 16S rDNA sequencing.ResultsResults were analyzed and compared based on milk pasteurization and source, spatial variability (core, outer, and under the rind), and length of aging (2–4 up to 12–18 months). The dominant organisms in the Gouda cheeses, based on percentage of sequence reads identified at the family or genus levels, were Bacillaceae, Lactococcus, Lactobacillus, Streptococcus, and Staphylococcus. More genus- or family-level (e.g. Bacillaceae) identifications were observed in the Gouda cheeses prepared with unpasteurized cow milk (120) compared with those prepared with pasteurized cow milk (92). When assessing influence of spatial variability on the metagenomics of the cheese, more pronounced differences in bacterial genera were observed in the samples taken under the rind; Brachybacterium, Pseudoalteromonas, Yersinia, Klebsiella, and Weissella were only detected in these samples. Lastly, the aging length of the cheese greatly influenced the number of organisms observed. Twenty-seven additional genus-level identifications were observed in Gouda cheese aged for 12–18 months compared with cheese only aged 2–4 months.ConclusionsCollectively, the results of this study are important in determining the typical microbiota associated with Gouda cheese and how the microbiome plays a role in safety and quality.

Methicillin-Resistant Staphylococcus aureus (MRSA) in Artisanal Cheeses in México.

Milk and dairy foods have frequently been implicated in staphylococcal food poisoning, and contaminated raw milk is often involved. The aim of the study was to determine the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in raw cow milk cheese produced in Mexico. A total of 78 unpasteurized cow milk cheese samples were screened for S. aureus. The isolates were identified as S. aureus based on morphology, Gram stain, catalase test, coagulase test, and mannitol salt agar fermentation. Isolates were subjected to biotyping, the methicillin resistance was analyzed using the disk diffusion, and the Staphylococcus enterotoxin A (SEA) production was examined by a dot-blot analysis. From a total of 78 samples of unpasteurized cheeses analyzed in this study, 44 cheeses were positive for S. aureus; however, a differential contamination between the different types of cheeses was observed, with high risk of contamination in adobero cheese (12, 95% CI 1.75 to 94.20; p=0.002). In this study, the frequency of methicillin-resistant Staphylococcus aureus (MRSA) was 18.1% (8/44) and of enterotoxin A producers was 18.1% (8/44). When classified by biotypes, MRSA only belongs to the human ecovar biotype (2/8, 25%) and the D biotype (4/8, 50%). S. aureus producers of enterotoxin A were distributed in specific nonhost biotypes.

Molecular detection of Salmonella spp., Listeria spp. and Brucella spp. in fresh artisanal cheese marketed in the city of Barranquilla: A pilot study

Introduction: Each year approximately 3 million people die as the result of foodborne diseases. The fresh artisan (handmade) cheese produced and distributed in the Colombian Caribbean region is a native product from the departments of Córdoba, Sucre, Bolívar, Atlántico, Magdalena, Cesar, and La Guajira. Its mass consumption increases the risk of infection with Salmonella spp., Listeria spp., and Brucella spp., as it is made with a very rustic technology, with unpasteurized cow milk, without standardized and hygienic procedures and its storage is inadequate. Objective: To detect the presence of Salmonella spp., Listeria spp., and Brucella spp. in samples of fresh artisan cheese from the Colombian Caribbean region. Materials and methods: Twenty-seven samples of cheese from five departments of the Caribbean Region (Atlántico (n=6), Bolívar (n=2), Córdoba (n=1), Magdalena (n=16), and Sucre (n=2)) were analyzed by real-time polymerase chain reaction (qPCR). Seventeen of the samples corresponded to soft cheese, five to semi-hard cheese and five to hard cheese. Results: In 62.9% (17/27) of the samples we detected Salmonella spp., in 70.4% (19/27), Listeria spp., and in 22.2% (6/27), Brucella spp., mainly from the department of Magdalena. In 62.5% (10/16) of the samples we detected Salmonella spp. and Listeria spp. while in the department of Atlántico, 50% (3/6) of the samples corresponded to Brucella spp. Conclusion: The results confirmed the presence of these microorganisms in all the samples of soft cheese from the Colombian Caribbean region.

Genome-Wide Identification of Fitness Factors in Mastitis-Associated Escherichia coli.

Virulence factors of mammary pathogenic Escherichia coli (MPEC) have not been identified, and it is not known how bacterial gene content influences the severity of mastitis. Here, we report a genome-wide identification of genes that contribute to fitness of MPEC under conditions relevant to the natural history of the disease. A highly virulent clinical isolate (M12) was identified that killed Galleria mellonella at low infectious doses and that replicated to high numbers in mouse mammary glands and spread to spleens. Genome sequencing was combined with transposon insertion site sequencing to identify MPEC genes that contribute to growth in unpasteurized whole milk, as well as during G. mellonella and mouse mastitis infections. These analyses show that strain M12 possesses a unique genomic island encoding a group III polysaccharide capsule that greatly enhances virulence in G. mellonella Several genes appear critical for MPEC survival in both G. mellonella and in mice, including those for nutrient-scavenging systems and resistance to cellular stress. Insertions in the ferric dicitrate receptor gene fecA caused significant fitness defects under all conditions (in milk, G. mellonella, and mice). This gene was highly expressed during growth in milk. Targeted deletion of fecA from strain M12 caused attenuation in G. mellonella larvae and reduced growth in unpasteurized cow's milk and lactating mouse mammary glands. Our results confirm that iron scavenging by the ferric dicitrate receptor, which is strongly associated with MPEC strains, is required for MPEC growth and may influence disease severity in mastitis infections.IMPORTANCE Mastitis caused by E. coli inflicts substantial burdens on the health and productivity of dairy animals. Strains causing mastitis may express genes that distinguish them from other E. coli strains and promote infection of mammary glands, but these have not been identified. Using a highly virulent strain, we employed genome-wide mutagenesis and sequencing to discover genes that contribute to mastitis. This extensive data set represents a screen for mastitis-associated E. coli fitness factors and provides the following contributions to the field: (i) global comparison of genes required for different aspects of mastitis infection, (ii) discovery of a unique capsule that contributes to virulence, and (iii) conclusive evidence for the crucial role of iron-scavenging systems in mastitis, particularly the ferric dicitrate transport system. Similar approaches applied to other mastitis-associated strains will uncover conserved targets for prevention or treatment and provide a better understanding of their relationship to other E. coli pathogens.

Identification and Antimicrobial Susceptibility of &amp;lt;i&amp;gt;Yersinia enterocolitica&amp;lt;/i&amp;gt; Found in Chitterlings, Raw Milk and Swine Fecal Samples

Foodborne illness is an escalating concern upon public health. The prevalence of Yersinia enterocolitica was assessed in chitterlings, raw milk and swine fecal from North Carolina. Uncleaned thirty chitterling samples procured from a local grocery store, forty-five swine fecal samples, and forty unpasteurized cow milk samples supplied by the University farm were evaluated for the presence of Y. enterocolitica. Isolates identified as presumptive positive were characterized as colonies with a pink or deep-red center on MacConkey and CIN agar, and verified further through polymerase chain reaction (PCR) for the presence of 16S rRNA gene for the Yersinia genera. Results showed that 4.4% swine fecal samples, 7.5% milk samples and 11.3% chitterling samples were presumptive positive for Y. enterocolitica by the direct plating method on selective agars. Of the thirty-chitterling samples examined by PCR for the 16S rRNA gene, 26% samples contained the identification gene for the bacteria of interest. After conducting virulence tests, the fecal samples were revealed as non-pathogenic. Only one of the milk samples were considered pathogenic and consisted of the following virulent genes: Yersinia heat-stable toxin (yst), invasion (inv), attachment invasion locus (ail), virulence regulon transcriptional activator (virF), Yersinia adehesin A (yadA), and the O:3 antigen gene (rfbC). Seven out of the eight (87.5%) chitterling samples were shown to be pathogenic. Disc diffusion was conducted to determine the antimicrobial susceptibility of the isolates. Over half (55.5%) of the antimicrobial agents were found effective, with isolates being completely susceptible to ciprofloxacin, kanamycin, trimethoprim, cefotaxime, and gentamycin. Ampicillin was determined to be least effective, where 84.6% of the samples presented resistance to the drug. Random amplified polymorphic DNA (RAPD) analysis and ERIC-PCR techniques were used to evaluate genetic similarity among the Yersinia isolates. There was approximately 85% similarity between two chitterlings and a fecal isolate during RAPD testing. With ERIC-PCR the largest similarity among all samples was at 95%, which was found between isolates from a chitterling and milk sample. Chitterling samples showed the highest prevalence of Y. enterocolitica compared to the other samples. Cross contamination at the farm level could be the root cause of this pathogen being prevalent in farm animal and food sources, which does pose a risk to public human health when food is improperly prepared.