Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

Background

A nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows was carried out on dairy farms in Sweden. The aim was to investigate the microbial panorama and the occurrence of antimicrobial resistance. Moreover, differences between newly infected cows and chronically infected cows were investigated.

Methods

In total, 583 quarter milk samples were collected from 583 dairy cows at 226 dairy farms from February 2008 to February 2009. The quarter milk samples were bacteriological investigated and scored using the California Mastitis Test. Staphylococci were tested for betalactamase production and presence of resistance was evaluated in all specific udder pathogens. Differences between newly infected cows and chronically infected cows were statistically investigated using logistic regression analysis.

Results

The most common isolates of 590 bacteriological diagnoses were Staphylococcus (S) aureus (19%) and coagulase-negative staphylococci (CNS; 16%) followed by Streptococcus (Str) dysgalactiae (9%), Str. uberis (8%), Escherichia (E.) coli (2.9%), and Streptococcus spp. (1.9%). Samples with no growth or contamination constituted 22% and 18% of the diagnoses, respectively. The distribution of the most commonly isolated bacteria considering only bacteriological positive samples were: S. aureus - 31%, CNS - 27%, Str. dysgalactiae - 15%, Str. uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1%. There was an increased risk of finding S. aureus, Str. uberis or Str. dysgalactiae in milk samples from chronically infected cows compared to findings in milk samples from newly infected cows. Four percent of the S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Overall, resistance to other antimicrobials than penicillin G was uncommon.

Conclusions

Staphylococcus aureus and CNS were the most frequently isolated pathogens and resistance to antimicrobials was rare.     

Microbiota of Cow’s Milk; Distinguishing Healthy,Sub-Clinically and Clinically Diseased Quarters

The objective of this study was to use pyrosequencing of the 16S rRNA genes to describe the microbial diversity of bovine milk samples derived from clinically unaffected quarters across a range of somatic cell counts (SCC) values or from clinical mastitis, culture negative quarters. The obtained microbiota profiles were used to distinguish healthy, subclinically and clinically affected quarters. Two dairy farms were used for the collection of milk samples. A total of 177 samples were used. Fifty samples derived from healthy, culture negative quarters with a SCC of less than 20,000 cells/ml (group 1); 34 samples derived from healthy, culture negative quarters, with a SCC ranging from 21,000 to 50,000 cells/ml (group 2); 26 samples derived from healthy, culture negative quarters with a SCC greater than 50,000 cells/ml (group 3); 34 samples derived from healthy, culture positive quarters, with a SCC greater than 400,000 (group 4, subclinical); and 33 samples derived from clinical mastitis, culture negative quarters (group 5, clinical). Bacterial DNA was isolated from these samples and the 16S rRNA genes were individually amplified and pyrosequenced. All samples analyzed revealed great microbial diversity. Four bacterial genera were present in every sample obtained from healthy quarters (Faecalibacterium spp., unclassified Lachnospiraceae, Propionibacterium spp. and Aeribacillus spp.). Discriminant analysis models showed that samples derived from healthy quarters were easily discriminated based on their microbiota profiles from samples derived from clinical mastitis, culture negative quarters; that was also the case for samples obtained from different farms. Staphylococcus spp. and Streptococcus spp. were among the most prevalent genera in all groups while a general multivariable linear model revealed that Sphingobacterium and Streptococcus prevalences were associated with increased 10 log SCC. Conversely, Nocardiodes and Paenibacillus were negatively correlated, and a higher percentage of the genera was associated with a lower 10 log SCC.     

Multiplex PCR assay for species identification of bovine mastitis pathogens

Aim: To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk. Methods and Results: A two‐tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10³ CFU ml^?1. A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples. Conclusion: The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. Significance and Impact of the Study: The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.     

Diagnosis of acute puerperal metritis by electronic nose device analysis of vaginal discharge in dairy cows

The objective of this study was to estimate the diagnostic accuracy of an electronic nose device using vaginal discharge samples to diagnose acute puerperal metritis (APM) in dairy cows. Uterine fluid was sampled manually with a gloved hand and under sterile conditions for electronic nose device analysis (day in milk (DIM) 2, 5, and 10) and bacteriologic examination (DIM 5), respectively, and on additional days, if APM was diagnosed during the daily clinical examinations. A dataset containing samples from 70 cows was used to create a model and to validate the APM status predicted by this model, respectively. Half of the dataset (n = 35; 14 healthy and 21 metritic cows) was provided with information regarding the APM diagnosis and contained all three measurements (DIM 2, 5, and 10) for each cow and was used as a training set whereas the second half was blinded (n = 35; 14 healthy and 21 metritic cows) and contained only the samples collected on DIM 5 of each cow and was used to validate the created prediction model. A receiver operating characteristic curve was calculated using the prediction results of the validation test. The best observed sensitivity was 100% with specificity of 91.6% when using a threshold value of 0.3. The calculated P-value for the receiver operating characteristic curve was less than 0.01. Overall, Escherichia coli was isolated in eight of 28 (28.6%) and 22 of 42 (52.4%) samples collected from healthy and metritic cows, respectively. Trueperella pyogenes and Fusobacterium necrophorum were isolated in 14 and six of 28 (50.0% and 21.4%) and 17 and 16 of 42 (40.5% and 38.1%) samples collected from healthy and metritic cows, respectively. The prevalence of Escherichia coli and Trueperella pyogenes was similar in the samples obtained from metritic cows used for the training set and the validation test. The results are promising especially because of the objective nature of the measurements obtained by the electronic nose device.     

Associations between intrauterine bacterial infection,reproductive tract inflammation,and reproductive performance in pasture-based dairy cows

Reproductive tract bacterial infections, particularly those caused by Escherichia coli and Trueperella pyogenes, can have a negative impact on reproductive performance. It has been hypothesized that the presence of E coli early postpartum may increase the risk of isolation of T pyogenes later postpartum. The objective of the present study was to examine associations between intrauterine bacterial infections with E coli and T pyogenes and any bacterial growth (irrespective of bacterial species), purulent vaginal discharge (PVD), cytologic evidence of endometritis (an increased proportion of polymorphonuclear cells [PMNs]), and reproductive performance. Dairy cows (n = 272) from six herds were examined at Days 0 (median, 2 days in milk), 21 and 42 postpartum. From each cow two intrauterine samples were collected via triple-guarded cytobrush at Days 0 and 21. The first cytobrush was used for bacteriologic culture. Escherichia coli and T pyogenes were isolated by culture, and E coli isolates were assigned to one of four phylogenetic groups using a two-step triplex polymerase chain reaction. In addition, T pyogenes was confirmed by polymerase chain reaction. The second cytobrush was used to prepare a cytology slide. Nucleated cells (n = 200) were categorized as epithelial cells, PMNs, or macrophages. Cows were also assessed for body condition score, PVD score, the presence of a CL, and pregnancy. Statistical analysis was performed using multivariable models. There was no association between the presence of E coli at Day 0 and probability of isolation of T pyogenes 3 weeks later; however, E coli positive cows at Day 0 were more likely to be diagnosed with E coli at Day 21 (relative risk [RR] = 2.0, P < 0.01). Escherichia coli at Day 0 or T pyogenes at Day 21 increased the risk of PVD diagnosis 3 weeks later (RR = 1.9; P = 0.04 and RR = 3.0; P = 0.05, respectively). Cows with any bacterial growth at Day 21, irrespective of species, were less likely to conceive within 3 weeks after the start of the seasonal breeding program (RR = 0.8; P = 0.05). Interestingly, cows with 25% PMNs or greater at Day 0 had shorter time to pregnancy (hazard ratio = 1.32; P = 0.05). Intrauterine bacterial infection may impair reproductive performance but the presence of E coli was not associated with isolation of T pyogenes 3 weeks later. Increased endometrial flux of PMNs in cows early postpartum may be a physiological process and improve reproductive performance.     

Prevalence of bacterial genotypes and outcome of bovine clinical mastitis due to <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> and <Emphasis Type="Italic">Streptococcus uberis</Emphasis>

Background

Streptococcus dysgalactiae and Streptococcus uberis are common causes of clinical mastitis (CM) in dairy cows. In the present study genotype variation of S. dysgalactiae and S. uberis was investigated, as well as the influence of bacterial species, or genotype within species, on the outcome of veterinary-treated CM (VTCM). Isolates of S. dysgalactiae (n = 132) and S. uberis (n = 97) were genotyped using pulsed-field gel electrophoresis. Identical banding patterns were called pulsotypes. Outcome measurements used were cow composite SCC, milk yield, additional registered VTCMs and culling rate during a four-month follow-up period.

Results

In total, 71?S. dysgalactiae pulsotypes were identified. Nineteen of the pulsotypes were isolated from more than one herd; the remaining pulsotypes were only found once each in the material. All S. uberis isolates were of different pulsotypes. During the follow-up period, the SCC of S. dysgalactiae-cows was significantly lower than the SCC of S. uberis-cows (P <0.05). Median SCC of S. dysgalactiae-cows was 71 500 cells/ml and of S. uberis-cows 108 000 cells/ml. No other differences in outcome parameters could be identified between species or genotypes.

Conclusions

Identical S. dysgalactiae genotypes were isolated from more than one herd, suggesting some spread of this pathogen between Swedish dairy herds. The genetic variation among S. uberis isolates was substantial, and we found no evidence of spread of this pathogen between herds. The milk SCC was lower during the follow-up period if S. dysgalactiae rather than S. uberis was isolated from the case, indicating differences in treatment response between bacterial species.

     

Profiling of the bacteria responsible for pyogenic liver abscess by 16S rRNA gene pyrosequencing

Pyogenic liver abscess (PLA) is a severe disease with considerable mortality and is often polymicrobial. Understanding the pathogens that cause PLA is the basis for PLA treatment. Here, we profiled the bacterial composition in PLA fluid by pyrosequencing the 16S ribosomal RNA (rRNA) gene based on next-generation sequencing (NGS) technology to identify etiological agents of PLA and to provide information of their 16S rRNA sequences for application to DNA-based techniques in the hospital. Twenty patients with PLA who underwent percutaneous catheter drainage, abscess culture, and blood culture for isolates were included. Genomic DNAs from abscess fluids were subjected to polymerase chain reaction and pyrosequencing of the 16S rRNA gene with a 454 GS Junior System. The abscess and blood cultures were positive in nine (45%) and four (20%) patients, respectively. Pyrosequencing of 16S rRNA gene showed that 90% of the PLA fluid samples contained single or multiple genera of known bacteria such as Klebsiella, Fusobacterium, Streptococcus, Bacteroides, Prevotella, Peptostreptococcus, unassigned Enterobacteriaceae, and Dialister. Klebsiella was predominantly found in the PLA fluid samples. All samples that carried unassigned bacteria had 26.8% reads on average. We demonstrated that the occurrence of PLA was associated with eight known bacterial genera as well as unassigned bacteria and that 16S rRNA gene sequencing was more useful than conventional culture methods for accurate identification of bacterial pathogens from PLA.     

Dynamics of bacteriologic and cytologic changes in the uterus of postpartum dairy cows

The objectives of this study were to characterize clinical, intrauterine, bacteriologic and cytologic changes during the first month after parturition in healthy dairy cows and in cows with subclinical endometritis (SE) or clinical endometritis (CE). Furthermore, risk factors related to clinical bacteriologic and cytologic findings were determined. A total of 170 calvings were enrolled, and intrauterine samples were collected on Days 0, 3, 9, 15, 21, and 28 postpartum using the cytobrush technique. The presence of Escherichia coli and Trueperella pyogenes was determined by Fourier transform infrared spectroscopy. The cows were categorized according to their uterine health status (UHS) on Day 21 as healthy (clear or absent vaginal discharge and <5% polymorphonuclear cells [PMN] in the cytologic sample), SE (clear or absent vaginal discharge and ≥5% PMN), or CE (vaginal mucus containing any signs of pus). The prevalence of SE and CE on Day 21 was 27.9% and 58.4%, respectively. Generally, samples from cows with SE and CE showed a greater bacterial growth density (BGD) than those from healthy cows. The BGD tended to be affected by the interaction of time by UHS (P = 0.057). Differences between healthy, SE, and CE cows were found from Day 3 to the last sampling day. Furthermore, the percentage of PMN differed between healthy, SE, and CE cows and was affected by time in a cubic way (decrease/increase/decrease). Overall, E coli was found in 25.4% of the samples, and T pyogenes was identified in 30.2% of the samples. The risk for CE was increased by BGD and the presence of T pyogenes. Conversely, the presence of E coli had no effect on the risk of CE or the risk of SE. The risk for an infection with T pyogenes was greater in the first-parity cows and in cows with assisted calving. In conclusion, changes in BGD and proportion of PMN varied with the UHS (healthy, SE, and CE), which was affected by the presence of T pyogenes but not E coli.     

Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.     

Prevalence of antibiotic resistant mastitis pathogens in dairy cows in Egypt and potential biological control agents produced from plant endophytic actinobacteria

Dairy production is threatened by antibiotic resistant pathogens worldwide, and alternative solutions to treat mastitis are not available. The prevalence of antibiotic resistant strains is not well known in less developed countries. The prevalence of pathogenic bacteria and their resistance to 21 commercial antibiotics were studied in milk samples taken from 122 dairy cows suffering from the symptoms of mastitis in Egypt. The bacterial species were identified with molecular methods, and antibiotic resistance was studied with disc diffusion method. The prevalence of Streptococcus aureus, Escherichia coli and Pseudomonas aeruginosa were 30%, 17% and 3.5%, respectively. Most (90%) of the S. aureus strains showed resistance to penicillin whereas only 10% of the strains were resistant to oxacillin. Nearly half (40%) of E. coli strains showed resistance to streptomycin. Six P. aeruginosa strains showed resistance to several antibiotics, including ceftriaxone, enrofloxacin and levofloxacin. This points out that despite P. aeruginosa was not common, it should be followed up carefully. Potential biocontrol agents against antibiotic resistant mastitis bacteria were searched among 30 endophytic actinobacterial strains derived from wild medicinal plants. Three plants, namely Mentha longifolia, Malva parviflora and Pulicaria undulata were chosen for a more detailed study; their endophytic actinobacteria were used to prepare metabolic extracts. The crude metabolites of the actinobacteria were extracted with ethyl acetate. All metabolic extracts inhibited the growth of S. aureus, methicillin-resistant Staphylococcus aureus (MRSA), E. coli and P. aeruginosa in vitro. The 16S rRNA sequence analysis revealed that the most efficient actinobacterial strains were two Micromonospora sp. and one Actinobacteria bacterium. We conclude that the combination of the metabolites of several endophytic actinobacteria derived from several medicinal plants would be the most efficient against pathogens. Different metabolite cocktails should be studied further in order to develop novel biocontrol agents to treat antibiotic resistant mastitis bacteria in dairy cows.     

Kill rate of mastitis pathogens by a combination of cefalexin and kanamycin

Aim: To assess the bacterial killing rate produced by a combination of cefalexin and kanamycin at two different concentration ratios. Methods and Results: Time–kill kinetics of cefalexin and kanamycin, individually and in combination, were determined against one strain each of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis. The combination was tested using two fixed ratios (cefalexin : kanamycin ratios of 1·25 : 1 and 1 : 2·3) and two concentrations of each ratio. Time–kill curves produced with either ratio were quite similar. Against most bacterial species, higher concentrations produced faster kill. In all cases, the combination of cefalexin and kanamycin showed faster and greater kill at lower antibiotic concentrations than those observed with either drug alone. Conclusions: The combination of cefalexin and kanamycin results in a fast initial killing of major mastitis pathogens at both concentration ratios. Significance and Impact of the Study: The combination of cefalexin and kanamycin achieved rapid bacterial kill at concentrations and ratios that can be achieved in vivo following intramammary infusion of a mastitis treatment.     

A clinical approach to determine false positive findings of clinical endometritis by vaginoscopy by the use of uterine bacteriology and cytology in dairy cows

Clinical endometritis in dairy cows is defined as mucopurulent or purulent vulvar discharge 21 days or more after parturition. The diagnosis of clinical endometritis is commonly based on vaginal examination. Techniques to reduce the proportions of false negative findings have been described. This paper discusses a clinical approach to determine the proportion of false positive findings that might occur by vaginal inspection. The consequences of false positive findings in dairy practice are unnecessary or inadequate treatments. In research, incorrect diagnoses have an impact on the interpretation of studies on the diagnosis and treatment of clinical endometritis. The objective of the present study was to compare intrauterine bacteriology and endometrial cytology in cows diagnosed with clinical endometritis with findings obtained by vaginoscopy. Clinical endometritis was defined as mucopurulent or purulent vulvar discharge. On two commercial dairy farms, cows were examined 21 to 28 d postpartum. Uterine samples (n = 230) were collected from cows with clinical endometritis with the cytobrush technique to determine the proportion of polymorphonuclear neutrophils (PMN) and to culture smears for aerobic bacteria. Two threshold values for the proportion of PMN (5 and 18%) were chosen as possible indicators for an inflamed endometrium. Common uterine pathogens A. pyogenes and E. coli were found in 33.5 and 10.4% of the samples, respectively. With increasing vaginal discharge score, proportion of samples positive for A. pyogenes increased significantly. The proportion of cows exceeding the thresholds for PMN increased with vaginal discharge score and the presence of A. pyogenes.Considering only the presence of aerobic uterine pathogens and a proportion of PMN above the threshold values of 5 and 18% as indicative for endometritis, a proportion of 17.3 and 28.5%, respectively, of diagnoses by vaginoscopy were false positive.     

Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> virulence genes in <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis> from Vellore,India

Streptococcus dysgalactiae subsp. equisimilis (SDSE), belonging to the group C and G streptococci, are human pathogens reported to cause clinical manifestations similar to infections caused by Streptococcus pyogenes. To scrutinize the distribution of gene coding for S. pyogenes virulence factors in SDSE, 255 isolates were collected from humans infected with SDSE in Vellore, a region in southern India, with high incidence of SDSE infections. Initial evaluation indicated SDSE isolates comprising of 82.35% group G and 17.64% group C. A multiplex PCR system was used to detect 21 gene encoding virulence-associated factors of S. pyogenes, like superantigens, DNases, proteinases, and other immune modulatory toxins. As validated by DNA sequencing of the PCR products, sequences homologous to speC, speG, speH, speI, speL, ssa and smeZ of the family of superantigen coding genes and for DNases like sdaD and sdc were detected in the SDSE collection. Furthermore, there was high abundance (48.12% in group G and 86.6% in group C SDSE) of scpA, the gene coding for C5a peptidase in these isolates. Higher abundance of S. pyogenes virulence factor genes was observed in SDSE of Lancefield group C as compared to group G, even though the incidence rates in former were lower. This study not only substantiates detection of S. pyogenes virulence factor genes in whole genome sequenced SDSE but also makes significant contribution towards the understanding of SDSE and its increasing virulence potential.     

Mediation analysis to estimate direct and indirect milk losses associated with bacterial load in bovine subclinical mammary infections

Milk losses associated with mastitis can be attributed to either effects of pathogens per se (i.e. direct losses) or to effects of the immune response triggered by the presence of mammary pathogens (i.e. indirect losses). Test-day milk somatic cell counts (SCC) and number of bacterial colony forming units (CFU) found in milk samples are putative measures of the level of immune response and of the bacterial load, respectively. Mediation models, in which one independent variable affects a second variable which, in turn, affects a third one, are conceivable models to estimate direct and indirect losses. Here, we evaluated the feasibility of a mediation model in which test-day SCC and milk were regressed toward bacterial CFU measured at three selected sampling dates, 1 week apart. We applied this method on cows free of clinical signs and with records on up to 3 test-days before and after the date of the first bacteriological samples. Most bacteriological cultures were negative (52.38%), others contained either staphylococci (23.08%), streptococci (9.16%), mixed bacteria (8.79%) or were contaminated (6.59%). Only losses mediated by an increase in SCC were significantly different from null. In cows with three consecutive bacteriological positive results, we estimated a decreased milk yield of 0.28 kg per day for each unit increase in log₂-transformed CFU that elicited one unit increase in log₂-transformed SCC. In cows with one or two bacteriological positive results, indirect milk loss was not significantly different from null although test-day milk decreased by 0.74 kg per day for each unit increase of log₂-transformed SCC. These results highlight the importance of milk losses that are mediated by an increase in SCC during mammary infection and the feasibility of decomposing total milk loss into its direct and indirect components.     

Potential Human Pathogenic Bacteria in a Mixed Urban Watershed as Revealed by Pyrosequencing

Current microbial source tracking (MST) methods for water depend on testing for fecal indicator bacterial counts or specific marker gene sequences to identify fecal contamination where potential human pathogenic bacteria could be present. In this study, we applied 454 high-throughput pyrosequencing to identify bacterial pathogen DNA sequences, including those not traditionally monitored by MST and correlated their abundances to specific sources of contamination such as urban runoff and agricultural runoff from concentrated animal feeding operations (CAFOs), recreation park area, waste-water treatment plants, and natural sites with little or no human activities. Samples for pyrosequencing were surface water, and sediment collected from 19 sites. A total of 12,959 16S rRNA gene sequences with average length of ≤400 bp were obtained, and were assigned to corresponding taxonomic ranks using ribosomal database project (RDP), Classifier and Greengenes databases. The percent of total potential pathogens were highest in urban runoff water (7.94%), agricultural runoff sediment (6.52%), and Prado Park sediment (6.00%), respectively. Although the numbers of DNA sequence tags from pyrosequencing were very high for the natural site, corresponding percent potential pathogens were very low (3.78–4.08%). Most of the potential pathogenic bacterial sequences identified were from three major phyla, namely, Proteobacteria, Bacteroidetes, and Firmicutes. The use of deep sequencing may provide improved and faster methods for the identification of pathogen sources in most watersheds so that better risk assessment methods may be developed to enhance public health.     

Risk factors for uterine diseases on small- and medium-sized dairy farms determined by clinical,bacteriological, and cytological examinations

The involution process of the postpartum bovine uterus is usually accompanied by invasion of various bacteria. The objectives of this study were to identify the relationship between the postpartum findings as risk factors for clinical endometritis (CE) and subclinical endometritis (SE). Furthermore, the effects of CE or SE on reproductive performance in small- and medium-sized dairy herds were investigated. A total of 400 cows were examined by vaginoscopy for CE at 20 to 30 days postpartum, and samples were collected for cytological examinations for SE and for bacteriology by cytobrush technique. The vaginoscopic and cytological examinations showed that 27.3% and 21.0% of the cows were found with CE and SE, respectively. The bacterial community analyses revealed a large variety of bacteria. Overall, bacteria from the order Actinomycetales, Lactobacillales, Bacillales, Burkholderiales, Caulobacteriales Enterobacteriales, Pasteurellales, and Pseudomonadales were detected, whereas in 39.5% of the samples no bacterial growth was detectable. The uterine pathogens Escherichia coli and Trueperella pyogenes were found in 16.8% and 13.0% of the samples cultivated under aerobic conditions. Other frequently isolated bacteria were Streptococcus spp. (31.3%), Staphylococcus spp. (20.0%), Corynebacterium spp. (16.5%), and Bacillus spp. (10.5%). The infection with T. pyogenes was the most important bacteriological risk factor for the occurrence of CE (odds ratio (OR) = 5.72; 95% CI = 3.07–10.83) and had a detrimental effect on the hazard of nonpregnancy by 200 days postpartum (hazard ratio = 1.66; 95% CI = 1.12–2.46). Calving assistance (OR = 1.79; 95% CI = 1.16–2.98) and farm (OR = 1.11; 95% CI = 1.02–1.20) were indicated as further risk factors for CE and SE. Effects of CE and SE on reproductive performance parameters could not be demonstrated.     

The effect of udder infection on the bacterial flora of the bulk milk of ten dairy herds

The significance of udder infection as a factor increasing the bacterial count of herd bulk milk was measured monthly for one year in ten dairy herds in Southern England. Staphylococcus aureus or mastitis streptococci were detected in 86% of samples, usually in numbers between 1000 and 10 000 c.f.u./ml of milk. However, in 8 and 2% of samples respectively > 20 000 or 100 000 c.f.u. of mastitis pathogens/ml of milk were detected. This occurred most commonly in the herds with a high incidence of Streptococcus uberis mastitis. The total bacterial counts of the herds' milks varied between 13 960 and 46 230 c.f.u./ml in the winter and between 6510 and 63 000 c.f.u./ml in the summer. No correlation was found between bacteriological quality of herd milk and the cleanliness of the milking machine and pipeline as assessed by plant rinses.     

Prevalence of bovine subclinical mastitis and isolation of its major causes in Bishoftu Town,Ethiopia

Objective

A cross-sectional study was conducted from November 2015 to March 2016 to estimate the prevalence, to assess the risk factors and to isolate the major etiological agent of subclinical mastitis in Bishoftu town. The study was conducted on 262 cross breed lactating cows selected from 12 intensively managed dairy farms. California mastitis test (CMT) and bacteriological culture methods were used as diagnostic tools.

Result

From 262 cows examined, 105 (40.1%) and from 1048 quarters examined, 170 (16.1%) were positive for sub-clinical mastitis using CMT. All CMT positive samples were cultured for etiological agent identification. From 170 samples cultured, 153 were positive for known subclinical mastitis pathogens. The dominant bacteria isolated were Staphylococcus species from these Staphylococcus aureus (44.9%) was the major one followed by Streptococcus spp. (25.3%) and other gram negative enteric bacteria, Escherichia coli (8.8%). Age, body condition score, milk yield, and number of parity were considered as potential risk factors; among these, age and number of parity have statistically significance association with the occurrence of subclinical mastitis (P < 0.05) both in the CMT and the bacteriological tests.

     

Ruminal Bacterial Community Composition in Dairy Cows Is Dynamic over the Course of Two Lactations and Correlates with Feed Efficiency

Fourteen Holstein cows of similar ages were monitored through their first two lactation cycles, during which ruminal solids and liquids, milk samples, production data, and feed consumption data were collected for each cow during early (76 to 82 days in milk [DIM]), middle (151 to 157 DIM), and late (251 to 257 DIM) lactation periods. The bacterial community of each ruminal sample was determined by sequencing the region from V6 to V8 of the 16S rRNA gene using 454 pyrosequencing. Gross feed efficiency (GFE) for each cow was calculated by dividing her energy-corrected milk by dry matter intake (ECM/DMI) for each period of both lactation cycles. Four pairs of cows were identified that differed in milk production efficiency, as defined by residual feed intake (RFI), at the same level of ECM production. The most abundant phyla detected for all cows were Bacteroidetes (49.42%), Firmicutes (39.32%), Proteobacteria (5.67%), and Tenericutes (2.17%), and the most abundant genera included Prevotella (40.15%), Butyrivibrio (2.38%), Ruminococcus (2.35%), Coprococcus (2.29%), and Succiniclasticum (2.28%). The bacterial microbiota between the first and second lactation cycles were highly similar, but with a significant correlation between total community composition by ruminal phase and specific bacteria whose relative sequence abundances displayed significant positive or negative correlation with GFE or RFI. These data suggest that the ruminal bacterial community is dynamic in terms of membership and diversity and that specific members are associated with high and low milk production efficiency over two lactation cycles.