The detection of obligate anaerobic bacteria in udder secretions of dry cattle with mastitis during summer: a comparison between gas-liquid chromatography and bacteriological culturing methods

Udder secretions sampled during the summer in 1984 and 1985 from mastitic quarters of 51 non-lactating cattle, mainly heifers less than 2 years of age, were examined bacteriologically for the presence of (facultative) aerobic and obligate anaerobic bacteria (OAB) and by gas-liquid chromatography (GLC) in order to detect volatile fatty acids (VFA), metabolic end-products of OAB. Forty-nine samples yielded positive cultures and in 20 cases these were mixtures of (facultative) aerobes and OAB. Only two specimens appeared to be sterile and from one specimen only were OAB cultured. Corynebacterium pyogenes was isolated from 35% of the cases and Peptococcus indolicus and Fusobacterium necrophorum from 31 and 22%, respectively. In most specimens (19/21) which yielded OAB after culturing, VFA (C3-C6) could be detected by GLC. Detection of VFA in summer mastitis secretions appeared to be a useful technique to evaluate the importance and association of OAB with summer mastitis. Because samples can be easily collected and stored at -20 degrees C, this is especially advantageous in situations where adequate facilities for the isolation of OAB are not readily available.     

Serodiagnosis of bovine paratuberculosis by use of a dot enzyme-linked immunosorbent assay

A dot ELISA was developed for detection of antibodies to Mycobacterium paratuberculosis. The assay was evaluated by testing sera from cattle that were determined, by bacteriologic culturing of feces, to be infected with M paratuberculosis and were suspected of having clinical disease. Further evaluation involved testing sera from cattle in which M paratuberculosis had not been isolated from feces on several attempts. Results of the dot ELISA were positive for sera from 86 of 101 infected cattle, and results were negative for sera from 64 of 64 noninfected cattle. Results of conventional ELISA and agar gel immunodiffusion (AGID) tests were positive for 79 of 99 and for 51 of 101 infected cattle, respectively. The dot ELISA also was evaluated by comparing results of testing 708 sera with results of bacteriologic culturing of matched fecal samples from 262 cattle in 3 central Ohio dairy herds known to include cattle infected with M paratuberculosis. Results of the dot ELISA were positive for 25 of 39 sera from cattle with positive results on culturing of concurrently obtained fecal specimens. The dot ELISA results were negative for 661 of 669 sera from cattle with negative results to culturing of concurrently obtained fecal specimens. The 39 sera from cattle with positive results on bacteriologic culturing of matched fecal specimens had positive results for ELISA and the AGID test 25 and 14 times, respectively. The 669 sera from cattle with concurrently negative results on bacteriologic culturing of feces had negative results to ELISA and the AGID test 559 and 668 times, respectively.     

瘤胃乙酸、丙酸比例对绵羊体内氧化代谢和血浆胰岛素水平动态变化的影响

本研究以全胃营养灌注绵羊为试验动物 ,用物质代谢区室分析方法和同位素示踪技术研究了瘤胃乙酸、丙酸比例对体内氧化代谢和血浆胰岛素水平动态变化的影响。不同试验处理间营养灌注液能量、蛋白质水平相同 ,而瘤胃灌注混合挥发性脂肪酸的乙酸、丙酸比例不同。三种混合挥发性脂肪酸的乙酸、丙酸比例分别为75 :15(VFA1)、65 :25(VFA2)、45 :45(VFA3)。由测定引入 14C -乙酸后血液二氧化碳放射比强度—时间曲线上升段斜率(k值)反映体内氧化代谢强度。试验结果为 ,瘤胃灌注VFA1 时的k值显著高于灌注VFA2 和VFA3 时的k值(P<0.05) ,而VFA2 和VFA3 两处理间的差异不显著(P>0.05) ,说明瘤胃灌注VFA1 时绵羊体内氧化代谢强度增加。向停止灌注24小时的绵羊瘤胃内灌注VFA1,血浆胰岛素水平在恢复灌注后缓慢上升。将瘤胃灌注混合挥发性脂肪酸由VFA1 换为VFA2 后 ,血浆胰岛素水平迅速上升 ,之后迅速下降 ,而由VFA2 换为VFA3 后未出现血浆胰岛素水平的明显变化 ,说明血浆胰岛素水平的变化与体内糖代谢调节状态有关 ,而瘤胃灌注VFA1 时的体内糖代谢调节状态与灌注VFA2 和VFA3 时明显不同。     

Isolation and identification of anaerobes in the veterinary diagnostic laboratory.

In a 1-year survey (1975 to 1976) of anaerobic bacteria recovered from diseased animals, anaerobes were as follows: Clostridium spp, 50%; gram-positive nonspore-forming anaerobic bacilli, 19%; gram-negative anaerobic bacilli, 19%; Actinomyces spp, 10%; and anaerobic cocci, 1%. Anaerobes were in approximately 61% of the specimens that were culturally positive for any bacteria. Approximately 25% of the specimens did not yield any bacteria (sterile specimens). The method for isolating and identifying anaerobes was based upon the use of reducible solid mediums and was specially designed for veterinary diagnostic laboratories. Emphasis was placed on the selection, collection, and transportation of specimens.     

The effect of diet on fecal moisture, osmolarity of fecal extracts, products of bacterial fermentation and loss of minerals in feces of weaned pigs

Litters of pigs were allotted to one of three dietary treatments. Treatment 1 (T1) consisted of a corn-soybean meal starter diet. Pigs fed treatment 2 (T2) received a steamed, rolled oat groats-casein diet and pigs in treatment 3 (T3) remained with the sow. Four pigs/treatment were used to investigate the difference in performance and the cause of post-weaning diarrhea associated with early weaning of pigs at 4 wk of age to a starter diet. Fecal moisture, osmolarity, acetic acid, lactic acid and glucose contents were all good indicators of dietary differences because of treatment X age interactions. These variables increased faster in fecal extracts from pigs fed the corn-soybean meal diet. Lactic acid, volatile fatty acid (VFA) levels, glucose and pH values were indicative of a more active bacterial fermentation in pigs receiving T1 than in those receiving T2 or pigs remaining with the sow (T3). Excess minerals appear to contribute significantly to the osmolarity of fecal material. Of the anions, lactate was the main contributor to the osmolarity of feces of T1 pigs, followed by P, VFA, of which acetic acid contributed 70%, and Cl. The main cations were K, Na and Ca. In T2, P was the main anion, followed by lactate, VFA and Cl, while the main cations were Na, K and Ca. Minerals seemed to be the major osmotic particles in fecal extracts of pigs remaining with the sow. Phosphorus was the major anionic contributor to osmolarity, followed by VFA, Cl and lactic acid. Potassium was the major cation, followed by Na and Ca.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of acids from inulin by a mixed culture of rumen microorganisms

The production of volatile fatty acids (VFA) and lactic acid from inulin (plant fructan with 2-1 bonds) in in vitro rumen fermentations was followed. The experiments were performed with inocula from wethers receiving two rations, with two pH regimes and using as inocula either whole rumen contents or the corresponding rumen fluid. The following results were obtained: The VFA production was higher when using inocula from hay and concentrate fed wethers. In contrast to this, the lactic acid production was higher when using inocula from hay fed wethers. The VFA production tends to be higher in a weakly acid medium than in a neutral medium. Acetate-to-propionate molar ratio was lower at lower pH. The amounts and composition of the VFA were not different when using both types of inocula. Thus, we believe that micro-organisms colonising plant fibres obviously do not play an important role in inulin splitting.     

Recovery and identification of anaerobes in veterinary medicine: A 2-year experience

The examination of 300 specimens over a 2-year period in a veterinary diagnostic laboratory (1975–1977) revealed that of the specimens yielding bacteria, 56.3% contained anaerobes while 71.8% yielded facultative bacteria. Of the total specimens 26.6% proved to be sterile and 4.6% were unsuitable. The anaerobes which were isolated with the highest frequency from diseased animals belonged to the following genera: Clostridium, 46.0%; Bacteroides 15.1%; Fusobacterium, 14.3%; Actinomyces, 11.1%; and Propionibacterium, 5.6%. The culture method used in this study was based upon reducible solid media for primary isolation. Its efficiency compared favorably with what is commonly accepted as an effective recovery rate for anaerobes. However, the relative proportion of anaerobic genera obtained from veterinary clinical specimens was in direct contrast with specimens obtained in medical diagnostic laboratories. Because of this difference and the kinds of anaerobes most frequently encountered in veterinary medicine, it is believed that the culture method used in this study is more suitable to veterinary medicine than other methods more commonly employed in medical diagnostic laboratories.     

Fecal D- and L-lactate, succinate and volatile fatty acid levels, and relationships with fecal acidity and diarrhea in neonatal calves

To clarify the significance of fecal organic anions in neonatal diarrhea, a total of 252 fecal samples (91 diarrheic, 161 normal) were collected from 136 dairy calves (including three crossbreds) less than 4 weeks old. Fecal pH, D‐ and L‐lactate, succinate and volatile fatty acid (VFA) were analyzed. In normal feces, lactate was highest and VFA was lowest at week 1 of age, and lactate progressively decreased and VFA progressively increased with advancing age. In diarrheic samples, although higher pH and lower lactate levels were confirmed at week 1, samples at weeks 3–4 showed lower pH and VFA accompanied by higher lactate of D and L‐isomers. In diarrhea, fecal butyrate was significantly lower at all stages, but succinate levels did not differ significantly. The proportion of lactate to organic anions (sum of lactate, succinate and VFA) in diarrheic feces was lower at week 1, and higher in weeks 2–4, while that of VFA to organic anions showed the opposite pattern. Strong relationships were observed between fecal pH and lactate, and VFA proportions in organic anions, though the relationship was weak in diarrhea. Most of the elevated lactate was observed in fecal samples with lower VFA. However, succinate had no relationship with VFA or lactate levels.     

Anaerobic bacterial infections and response to treatment in dogs and cats: 36 cases (1983-1985)

Anaerobic bacteria have been increasingly implicated as important pathogens in animals. To determine the prevalence of anaerobic bacterial infection, the results of anaerobic bacteriologic culture of 599 specimens obtained from dogs and cats hospitalized at the Colorado State University Veterinary Teaching Hospital were reviewed. Obligate anaerobic bacteria were isolated from 35% of properly submitted specimens; Bacteroides spp and Fusobacterium spp were the organisms most commonly isolated. Infections most often containing anaerobes were abscesses, pleuropulmonary infections, and abdominal infections. A complication rate of 28% was observed with anaerobic bacterial infections; failure to initially treat with an effective antibiotic increased the rate of recurrence of infection.     

Effect of ruminal infusion of glucose, volatile fatty acids and hydrochloric acid on mineral metabolism in sheep

Two experiments were conducted to study the effects of alterations in ruminal pH and volatile fatty acid (VFA) concentrations on utilization of Mg and other minerals. In Exp. 1, two metabolism trials were conducted with 12 ruminally cannulated crossbred wethers fed 800 g/d of orchard-grass (Dactylis glomerata, L.) hay. After each feeding, wethers were ruminally infused with 500 ml (4.2 ml/min) or either 1) deionized water, 2) 40% (w/v) glucose solution, 3) .26 M propionic and .17 M butyric acid solution or 4) .35 M HCl. The pH of the VFA solution was adjusted to 6.8 with 10N NaOH. In Exp. 2, a metabolism trial was conducted with 12 ruminally cannulated crossbred wethers fed 600 g of orchard-grass hay and infused with a buffered VFA solution prepared as in Exp. 1 or with an unbuffered solution. In both experiments each trial consisted of a 5-d adaption period followed by four 5-d collections of feed, feces and urine. Compared with the glucose treatment, infusion of the buffered VFA solution produced similar acetic and propionic and higher (P less than .05) butyric acid concentrations (Exp. 1). The HCl solution produced changes in ruminal and pH values similar to those of the glucose infusion. In Exp. 1, apparent absorption of Mg was increased over twofold by the glucose infusion (P less than .05), but the other infusions had no effect. Apparent absorption of P was decreased (P less than .05) by HCl infusion, and K absorption was decreased by HCl and glucose infusions. In Exp. 2, infusion of the unbuffered VFA solution decreased apparent Mg absorption by 15.7%, compared with infusion of the buffered solution. These experiments suggest that the increased Mg absorption observed with carbohydrate supplementation is not due to alterations in ruminal pH or VFA levels.     

BSAVA EDUCATION COMMITTEE COMMISSIONED ARTICLE: Anaerobic bacteria: a cause of infection?

This review article considers the evidence available for a significant role of obligate anaerobes in infections of small animals. The presence of anaerobes in the normal flora and the meaning of the terms aerobe and anaerobe both in relation to the bacterial metabolism and laboratory culture is discussed. The reported findings of anaerobes in clinical veterinary conditions are reviewed. The particular importance of the non-sporing (non-clostridial) anaerobes in traumatic and endogenous infections and the mixed nature, both in terms of different anaerobes and mixed aerobes and anaerobes of such infection are considered. Some experimental evidence for the pathogenicity of obligate anaerobes and their complex role in mixed infections is given. Observations on the use of antibiotics for treatment of suspected anaerobic infections are discussed and the possible implications to laboratory procedures for antibiotic sensitivity testing.     

Anaerobic bacteria in 21 horses with pleuropneumonia

Anaerobic bacteria are important and overlooked bacterial pathogens of the lower respiratory tract in horses. Twenty-one of 46 horses with pleuropneumonia had anaerobic bacteria isolated from pleural fluid or from tracheobronchial aspirate. Bacteroides oralis and B melaninogenicus were the anaerobes most frequently isolated. Survival was significantly less for horses from which anaerobes were isolated than for horses from which anaerobes were not isolated. Putrid odor was associated with the pleural fluid and/or breath in 62% of the horses from which anaerobes were isolated. In these horses, the survival rate was significantly less than for horses from which odoriferous specimens were not isolated.     

Factors affecting the in vitro production of volatile fatty acids by mixed bacterial populations from the bovine rumen

Factors affecting in vitro ruminal bacterial VFA production were examined. Treatments consisted of high and low initial pH (6.7, 5.7), osmolality (600, 400 mOsm) and concentrations of acetic (40, 0 mM) and propionic acids (20, 0 mM). Response variables measured included the production of acetic, propionic and total VFA, total gas and methane. Initial pH affected (P less than .05) most variables either independently or in combination with one or more of the other factors. Acetic acid production was reduced 40% (P = .03) when initial acetic acid concentrations were 40 mM compared with 0 mM. Also, acetic acid production was less (P less than .01) at low initial pH (5.7) than at high initial pH (6.7). Propionic acid production was greater (P = .05) at high vs low initial acetic acid concentrations. Propionic acid production was greater in response to low vs high initial osmolality, although the magnitude of this difference depended on initial pH (interaction P = .02). Total production of VFA was greater (P less than .01) at high than at low initial pH; however, at low initial pH, no difference (P greater than .05) was observed due to initial osmolality, whereas at high pH, production was greater (interaction P = .04) for low than for high initial osmolality. The diminished production of total VFA at pH 5.7 occurred primarily due to reduced acetic acid production, although increased production of propionic and butyric acids was noted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of coliform bacteria, feed deprivation, and pH on ruminal D-lactic acid production by steer or continuous-culture microbial populations changed from forage to concentrates

Fecal coliform bacteria were isolated from three herbivores (cattle, horse, and red panda) and shown to produce primarily the D-form of lactate, plus acetate and ethanol when grown anaerobically in 1.0% glucose broth. To evaluate coliform contribution to D-lactate acidosis in cattle, experiments involving a forage-adapted steer (fasted or normally fed) and four 500-ml fermentors were compared during 3 d of grain overload. In both systems, coliforms and D- and L-lactic acid production were greater from fasted than from normally fed steer inoculum. With fasted inoculum, coliform counts peaked (3 x 10(7)/ml at 7 h after initial engorgement) and receded to 10(3)/ml by the time D-lactate concentration peaked, indicating that bacteria other than coliform were responsible for the delayed peaking of D- (48 h) compared with L-lactate (24 h). Increases in lactobacilli more closely mimicked D-lactate increases than did changes in coliforms. The comparisons between the steer and fermentors showed many similar shifts in end-products and groups of bacteria, more so with the experiment initiated with fasted than with normal inoculum. With normal inoculum, VFA content and moles of butyrate/100 mol of VFA were greater in vitro than in vivo; VFA content presumably was larger because of VFA absorption in vivo. In a separate experiment, cultures initiated with identical inoculum and given the same amount of feed accumulated more lactate when pH was permitted to decrease to 5.0 than when pH was maintained at 5.5 for 6.0 or above, indicating the role buffers can have in controlling acidosis during diet change to concentrates.     

Improved detection of Tritrichomonas foetus in bovine diagnostic specimens using a novel probe-based real time PCR assay

A Tritrichomonas foetus-specific 5' Taq nuclease assay using a 3' minor groove binder-DNA probe (TaqMan MGB) targeting conserved regions of the internal transcribed spacer-1 (ITS-1) was developed and compared to established diagnostic procedures. Specificity of the assay was evaluated using bovine venereal microflora and a range of related trichomonad species. Assay sensitivity was evaluated with log(10) dilutions of known numbers of cells, and compared to that for microscopy following culture (InPouch TF test kit) and the conventional TFR3-TFR4 PCR assay. The 5' Taq nuclease assay detected a single cell per assay from smegma or mucus which was 2500-fold or 250-fold more sensitive than microscopy following selective culture from smegma or mucus respectively, and 500-fold more sensitive than culture followed by conventional PCR assay. The sensitivity of the conventional PCR assay was comparable to the 5' Taq nuclease assay when testing purified DNA extracted from clinical specimens, whereas the 5' Taq nuclease assay sensitivity improved using crude cell lysates, which were not suitable as template for the conventional PCR assay. Urine was evaluated as a diagnostic specimen providing improved and equivalent levels of T. foetus detection in spiked urine by both microscopy following culture and direct 5' Taq nuclease detection, respectively, compared with smegma and mucus, however inconclusive results were obtained with urine samples from the field study. Diagnostic specimens (n=159) were collected from herds with culture positive animals and of the 14 animals positive by 5' Taq nuclease assay, 3 were confirmed by selective culture/microscopy detection (Fisher's exact test P<0.001). The 5' Taq nuclease assay described here demonstrated superior sensitivity to traditional culture/microscopy and offers advantages over the application of conventional PCR for the detection of T. foetus in clinical samples.     

Simultaneous detection and strain differentiation of Mycobacterium bovis directly from bovine tissue specimens by spoligotyping

Culture of Mycobacterium bovis is used routinely to support field diagnosis of bovine tuberculosis; however, this method is slow. Rapid detection and strain-typing of M. bovis directly from 37 lesioned bovine lymph node specimens was performed by the polymerase chain reaction (PCR) based method, spoligotyping. Mycobacterial DNA was extracted from the specimens using a nucleic acid sequence capture technique. Two sets of specimens were tested, the first set comprising 16 decontaminated tissue homogenates from lesioned lymph node specimens which had been processed for BACTEC culture and a second set of 21 non-decontaminated lesioned lymph node specimens. Both sets of specimens had been frozen before analysis. Sequence capture PCR enabled detection and strain-typing of M. bovis directly from 15 of the 16 decontaminated homogenates and all 21 of the non-decontaminated tissues. Four spoligotype (ST) patterns were obtained from each set; ST1, ST2, ST3 and ST16 were detected in the decontaminated specimens and ST1, ST2, ST11 and ST14 in the non-decontaminated specimens. For both sets of specimens, ST1 was the predominant strain type detected. ST patterns obtained from the BACTEC cultures of the decontaminated specimens were in agreement with those obtained directly from the tissue. The sensitivity of detection by sequence capture-PCR compared very favourably with that of BACTEC culture. ST patterns were obtained directly from tissues of 34 of the 35 culture positive specimens and the two culture negative specimens. DNA extraction from the 21 non-decontaminated specimens involved an initial stomaching treatment. An assessment of sequence capture on both liquid alone and liquid and tissue homogenate combined, following stomaching, indicated that PCR was less successful on the liquid component alone.     

Pre-harvest factors influencing the acid resistance of Escherichia coli and E. coli O157:H7

The effects of pH, acetate, propionate, or butyrate concentration, and diet on acid resistance of fecal Escherichia coli and E. coli O157:H7 were determined by in vitro and in vivo experiments. The pH tested was from 4.0 to 8.0, and the VFA concentrations tested were 0 to 100 mM. The E. coli O157:H7 used was strain 505B. In an in vivo study, cattle were fed a grain-based diet, then either not switched or switched to a grain-based diet with 3% added calcium carbonate or two fiber-based diets (soybean hulls or hay). Acid resistance was expressed as viability after acid-shock at pH 2.0 for 1 h and 4 h for fecal E. coli and E. coli O157:H7, respectively. Enumeration methods used were multitube fermentation, agar plate, and petri-film methods. The E. coli O157:H7 was not found in continuous culture inocula or in vivo samples. The viability of fecal E. coli decreased linearly (P < 0.01) as the culture pH increased, and viability of E. coli O157:H7 was highest (P < 0.01) when cultivated at pH 6.0. The viability of fecal E. coli and E. coli O157:H7 showed quadratic responses (P < 0.05) as acetate and butyrate concentrations increased at pH 7.2, with maximal acid resistance at 20 and 12 mM, respectively. As propionate concentration increased, the acid resistance was not different (P > 0.05) for fecal E. coli. Acid resistance of E. coli was induced by acetate and butyrate, even though the environmental pH was near neutral. Similar results were measured in the in vivo study, where viability after acid shock was more dependent on VFA concentration than on pH. Increasing the dietary calcium carbonate concentration also increased (P < 0.05) acid resistance of fecal E. coli. Results from these studies demonstrated that culture pH and VFA affect acid resistance of E. coli.     

In situ hybridization for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods.

Archived formalin-fixed, paraffin-embedded tissues from 25 pigs naturally infected with transmissible gastroenteritis virus (TGEV) were examined by in situ hybridization for TGEV nucleic acid using a nonradioactive digoxigenin-labeled cDNA probe that targeted the nucleocapsid sequence of TGEV strains. The results of in situ hybridization for the detection of TGEV were compared with virus isolation (VI), a fluorescent antibody test (FAT), and transmission electron microscopy (TEM). VI, FAT, and TEM were tested over a course of time before the in situ hybridization was performed. Positive hybridization signals were detected in duodenal, jejunal, and ileal enterocytes from 21 pigs. Hybridization signals were confined to the cytoplasm. Intestinal specimens from 25 piglets were evaluated by 4 tests. Twenty-one of 25 were positive by in situ hybridization. Of these 21 samples, 5 (24%) were positive for TGEV by all 4 tests, 15 (71%) were positive by FAT, 14 (67%) were positive by VI, and 6 (29%) were positive by TEM. In situ hybridization for the detection of TGEV in formalin-fixed, paraffin-embedded tissues provides a rapid means of confirmation of a histopathological diagnosis of TGEV without virus isolation, or when only formalin-fixed intestinal specimens were available.     

Relative sensitivity of polymerase chain reaction assays used for detection of feline herpesvirus type 1 DNA in clinical samples and commercial vaccines

OBJECTIVE: To determine relative detection rates and detection limits for 6 published polymerase chain reaction (PCR) assays used for detection of feline herpesvirus type 1 (FHV-1) DNA. SAMPLE POPULATION: 5 vaccines licensed for use in preventing FHV-1-associated disease; 15 conjunctival biopsy specimens collected from cats with keratitis, conjunctivitis, or both; and a plaque-purified field isolate of FHV-1 cultured in vitro. PROCEDURE: Vaccines and clinical samples were assessed for FHV-1 DNA by use of all 6 assays. Detection rates were calculated by assuming that any sample in which FHV-1 DNA was detected was a true-positive result. Detection limits were estimated by use of serial dilutions of DNA extracted from cultured FHV-1 and 1 clinical sample. RESULTS: Testing by use of all 6 assays resulted in detection of FHV-1 DNA in all 5 vaccines. Testing by use of all 6 assays yielded concordant results for 9 of 15 conjunctival biopsy specimens (8 with negative results and 1 with a positive result). Calculated detection rates for clinical samples ranged from 29% to 86%. Assay sensitivity was ranked similarly by use of detection rate or detection limit. CONCLUSIONS AND CLINICAL RELEVANCE: Testing by use of all assays was equally likely to detect vaccine virus. Therefore, a positive PCR result in a cat may reflect vaccine virus rather than wild-type virus. Test sensitivity as assessed by detection limits and detection rates varied greatly. Because FHV-1 can be shed in clinically normal animals, high detection rate will not necessarily correlate with high diagnostic sensitivity.