Plasma prolidase activity as a possible diagnostic index of chronic hepatic abscess in cattle

The usefulness of prolidase as a biochemical parameter to represent the chronic state of hepatic abscess was discussed in eight cattle experimentally inoculated with Fusobacterium necrophorum and 18 spontaneously affected cattle. Blood was daily collected to measure the plasma prolidase activity and sialic acid level from the experimental cattle for 90 days after inoculation. In three out of four cattle affected with hepatic abscess, prolidase activity began to rise about 40 days after inoculation, and maintained high activity till 90 days. In the same cattle the sialic acid concentration increased from 7 to 10 days after inoculation, and gradually returned to the normal value 50 days after it. Another cow showed a similar change in early stage of experiment, but prolidase activity decreased after 70 days and sialic acid concentration maintained high level till 90 days. In two cattle, which showed scars but no abscess on autopsy, the prolidase activity increased temporarily from 40 to 55 days after inoculation. In the control cattle inoculated with an inactivated bacterial suspension, neither the sialic acid level nor the prolidase activity showed any large variation in the experimental period. Among the spontaneously affected cattle, those with a high sialic acid level revealed normal prolidase activity and those with a normal sialic acid level had high prolidase activity.     

Determinants and Utility of the Anion Gap in Predicting Hyperlactatemia in Cattle

The objectives of this study were to investigate the determinants of the anion gap (AG) in cattle and to evaluate the utility of AG in detecting hyperlactatemia in sick neonatal calves and adult cattle. The AG was calculated as AG = ([Na+] + [K+]) - ([Cl^-] + [HCO₃]), with all values in mEq/L. The AG of healthy neonatal calves (n = 16) was 29.6 ± 6.2 mEq/L (mean ± SD), and the blood L-lactate concentration ranged from 0.5 to 1.2 mM/L. The AG was significantly (P > .05) correlated with serum phosphate (r = .66) and creatinine (r = .51) concentrations. The AG of neonatal calves with experimentally induced diarrhea (n = 16) was 28.6 ± 5.6 mEq/L, and the blood L-lactate concentration ranged from 1.1 to 2.9 mM/L. The AG was significantly correlated with blood L-lactate concentration (r = .67), serum phosphate concentration (r = .63), creatinine concentration (r = .76), and blood pH (r = -.61). The AG of adult cattle with abomasal volvulus (n = 41) was 20.5 ± 7.8 mEq/L, and the blood L-lactate concentration ranged from 0.6 to 15.6 mM/L. The AG was significantly correlated with blood L-lactate concentration (r = .60), serum phosphate concentration (r = .71), creatinine concentration (r = .65), albumin concentration (r = .47), total protein concentration (r = .54), blood pyruvate concentration (r = .67), and blood pH (r = -.41) but not plasma β-OH butyrate concentration. The results indicate that the AG in cattle is only moderately correlated with blood L-lactate concentration and is similarly correlated with serum phosphate and creatinine concentrations in neonatal calves and adult cattle, as well as with serum albumin and total protein concentrations in adult cattle. Anion gap determination is of limited usefulness in predicting blood L-lactate concentration in sick cattle, whereas the correlation between AG and serum creatinine concentration in sick cattle suggests that an increased AG should alert the clinician to the potential presence of uremic anions.     

Hepatic ultrasonography and blood changes in cattle with experimentally induced hepatic abscesses.

Hepatic abscesses were induced experimentally in 5 steers by inoculating Fusobacterium necrophorum via ultrasonography-guided, percutaneous catheterization of the portal vein. Hepatic ultrasonography was performed to determine the onset and progression of abscessation. Blood samples were collected before and after inoculation for performing leukocyte counts and hepatic function tests. Ultrasonographic evidence of liver abscesses was observed as early as 3 days after inoculation. Abscesses appeared as hyperechoic centers (cellular debris and pus) surrounded by hypoechoic or anechoic areas (fluid). Increases in rectal temperature, leukocyte counts, fibrinogen, globulin, bilirubin, gamma-glutamyltransferase, and sorbitol dehydrogenase concentrations were detected. Hepatic dysfunction was evidenced by decrease in serum albumin concentration and low sulfobromophthalein clearance. The ultrasonographic diagnosis of abscesses correlated well with necropsy findings.     

Bacterial flora of liver abscesses in feedlot cattle fed tylosin or no tylosin.

Bacterial flora of liver abscesses from cattle fed tylosin or no tylosin and susceptibilities of the predominant bacterial isolates to tylosin and other antimicrobial compounds were determined. Abscessed livers were collected at slaughter from cattle originating from feedlots that had fed tylosin (n = 36) or no tylosin (n = 41) for at least 2 yr, and segments of livers with one or two intact abscesses were transported to the laboratory. Abscesses were cultured for anaerobic and facultative bacteria. Fusobacterium necrophorum, either as single culture or mixed with other bacteria, was isolated from all abscesses. The incidence of subsp. necrophorum, as part of the mixed infection, was lower (P < .05) in the tylosin group than in the no-tylosin group (33 vs 61%). However, the incidence of Actinomyces pyogenes was higher (P < .01) in the tylosin group than in the no-tylosin group (53 vs 10%). Totals of 119 F. necrophorum and 21 A. pyogenes isolates were used for determinations of susceptibilities to bacitracin, oxytetracycline, chlortetracycline, lasalocid, monensin, tylosin, tilmicosin, and virginiamycin. The minimum inhibitory concentrations (MIC) of antibiotics were determined with a broth microdilution method. The mean MIC of tylosin for F. necrophorum and A. pyogenes were not different between isolates from tylosin and no-tylosin groups. We concluded that continuous feeding of tylosin did not induce resistance in F. necrophorum or A. pyogenes. Also, the higher incidence of mixed infection of F. necrophorum and A. pyogenes in liver abscesses of tylosin-fed cattle suggests a potential synergistic interaction between the two organisms in causing liver abscesses.     

Experimental infection of calves with epizootic hemorrhagic disease virus.

OBJECTIVE: To determine whether experimental inoculation with a field strain of epizootic hemorrhagic disease virus serotype-2 (EHDV-2) suspected of causing clinical disease in naturally infected cattle would cause clinical disease in calves. ANIMALS: 8 calves. PROCEDURE: A strain of EHDV-2 isolated from a white-tailed deer that died of hemorrhagic disease was passaged twice in deer and used to inoculate 6 calves SC and ID; the other 2 calves were used as controls. Physical examinations, CBC, lymphocyte blastogenesis assays, and coagulation assays were performed; rectal temperature, interferon production, and serum neutralizing antibody responses were measured; and virus isolation was attempted every other day for 21 days after inoculation and then every fourth day for another 30 days. Calves were euthanatized on postinoculation day 51, and necropsy was performed. RESULTS: Calves inoculated with EHDV-2 became infected, as evidenced by development of viremia and seroconversion. However, the virus did not cause detectable clinical disease, clinicopathologic abnormalities, or gross lesions. Viremia was prolonged despite development of a serum neutralizing antibody response. A white-tailed deer inoculated with the same EHDV-2 strain developed clinical signs of epizootic hemorrhagic disease, demonstrating that the inoculum was virulent. CONCLUSION: Calves experimentally infected with EHDV-2 developed viremia and seroconverted but did not develop detectable clinical disease.     

Isolation, characterization, and quantitative measurement of serum alpha 1-acid glycoprotein in cattle

Bovine alpha 1-acid glycoprotein (alpha 1AG) was purified from pooled normal bovine sera by successive ammonium sulfate precipitation, ion-exchange chromatographies and gel filtration. Bovine alpha 1AG had a molecular weight of 42,000 +/- 2,000 and a sedimentation coefficient of 3.4S. It contained 26.6% carbohydrate. Gel isoelectric focusing revealed a microheterogeneity with 7 to 8 bands in a pI range of 3.2 to 3.7. It migrated to the alpha 1-globulin region upon immunoelectrophoresis. Single radial immunodiffusion was developed for the quantitative measurement of bovine alpha 1AG in serum. The mean serum value of alpha 1AG in 152 healthy Holstein cattle (1-12 years old) was 283.2 +/- 82.3 micrograms/ml. Elevated values (cut-off value = 450 micrograms/ml) were observed in cattle with traumatic pericarditis (100%), arthritis (100%), mastitis (91%), pneumonia (70%), and mesenteric liponecrosis (43%).     

Liver abscesses in Holstein dairy cattle: 18 cases (1992-2003)

BACKGROUND: The characteristic clinical manifestations, clinicopathologic findings, treatment, and outcome of dairy cattle with liver abscess are poorly defined. ANIMALS: The study included 18 Holstein cows with liver abscesses. METHODS: A retrospective study of medical records was performed. Cattle with liver abscess were identified by ultrasound examination or exploratory laparotomy. RESULTS: The most common reason for examination was anorexia (14/18). Five cows had fever, 5 were bradycardic, and 5 were tachycardic. Peritonitis (n=6) and vagal indigestion (n=4) were the most frequently associated diseases. Neutrophilia (n=14), hyperfibrinogenemia (n=11), and high serum globulin concentration (n=10) were characteristic of chronic inflammation. Evidence of liver disease on serum biochemistry profile was uncommon. The most common bacterium isolated from the abscess was Arcanobacterium pyogenes (n=4). Anaerobic bacteria were isolated frequently (n=7). There were 6 polymicrobial isolates, with both aerobic and anaerobic bacteria, out of 8 positive samples. Medical treatment was successful in 5 of 7 cattle. Five cows were euthanized and postmortem examination revealed 2 cattle with thrombosis of caudal vena cava. CONCLUSIONS AND CLINICAL IMPORTANCE: Holstein dairy cattle affected by liver abscess exhibit no pathognomonic clinical signs. Clinicopathologic findings were often consistent with a chronic active inflammation. Liver abscesses should be included in the differential diagnosis in cattle with a chronic inflammatory process, cranial peritonitis, or vagal indigestion. Prolonged treatment with antimicrobials might be successful.     

Effects of tylosin on concentrations of Fusobacterium necrophorum and fermentation products in the rumen of cattle fed a high-concentrate diet.

OBJECTIVE: To determine effects of tylosin on ruminal concentrations of Fusobacterium necrophorum and fermentation products in cattle during rapid adaptation to a high-concentrate diet. ANIMALS: 6 steers fitted with ruminal cannulas. PROCEDURE: Steers were assigned randomly to 2 treatment groups and switched from a 0 to an 85% concentrate diet during a 4-day period. Cattle received this diet, with or without tylosin (90 mg/steer/d), for 4 weeks. Samples of ruminal contents were collected daily beginning 2 days before the treatment protocol and in the first week of concentrate feeding. Four subsequent samples were collected at weekly intervals. Concentration of F. necrophorum in samples was determined, using the most-probable-number technique. Ruminal pH and concentrations of volatile fatty acids (VFA), lactate, and ammonia also were determined. All steers received both treatments separated by 4 weeks (cross-over design), during which time they were fed alfalfa hay only. RESULTS: In control steers, concentration of F. necrophorum increased in response to the high-concentrate diet. Tylosin-fed steers had lower concentrations of F. necrophorum than control steers at all times during concentrate feeding. However, ruminal pH and concentrations of lactate, VFA, and ammonia did not differ between treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Tylosin caused a significant reduction in ruminal concentrations of F. necrophorum during rapid adaptation to a high-concentrate diet but had no effect on fermentation products. The reduction in ruminal concentration of F. necrophorum helps explain the reduction in prevalence of hepatic abscesses reported in tylosin-fed feedlot cattle.     

Bacteriologic and histologic studies of hepatic abscesses in cattle

Twenty-eight abscessed livers were collected from feedlot cattle at an abattoir; specimens were obtained from 49 abscesses for bacteriologic culture and for histologic examination. Cultural procedures included techniques to enumerate and isolate facultative and obligate anaerobic bacteria. Anaerobic bacteria were isolated from all 49 abscesses, whereas facultative bacteria were isolated from only 22. Mean bacterial counts for anaerobic and facultative bacteria were 3 X 10(8) and 8 X 10(8) bacteria/g of purulent material, respectively. Fusobacterium necrophorum, the only anaerobe isolated, was detected in 100% of the abscesses. Fusobacterium necrophorum biotype A was isolated from 57% of the abscesses (in pure culture from 75%), and F necrophorum biotype B was isolated from 47% of the abscesses (from 96% with mixed infections). Corynebacterium pyogenes was the predominant facultative bacterium isolated. Histologic changes in abscesses were qualitatively similar; abscesses were pyogranulomatous, with a necrotic center surrounded by zones of inflammatory tissue. However, the severity of lesions varied, depending on the F necrophorum biotype involved. Portal triad fibrosis and bile-duct proliferation were most severe in biotype A and mixed biotype B infections and less severe in abscesses from which biotype B was isolated in pure culture.     

Comparative virulence of isolates of bovine viral diarrhea virus type II in experimentally inoculated six- to nine-month-old calves

OBJECTIVE: To determine the comparative virulence of 5 isolates of bovine viral diarrhea virus (BVDV) type II by inoculating 6- to 9-month-old beef calves with isolates originating from the tissues of cattle affected with naturally occurring, transient, acute, nonfatal infections or naturally occurring, peracute, fatal infections. ANIMALS: 22 calves that were 6 to 9 months old. PROCEDURE: The study used BVDV isolates 17011, 713, and 5521 that originated from fetuses aborted from cows with transient, nonfatal, acute BVDV infections and isolates 23025 and 17583 that originated from the tissues of cattle with peracute, fatal BVDV infections. Calves were allotted to 6 groups (1, mock-infected control calves [n = 2]; 2, inoculated with BVDV 17011 [4]; 3, inoculated with BVDV 713 [4]; 4, inoculated with BVDV 5521 [4]; 5, inoculated with BVDV 23025 [4]; and 6, inoculated with BVDV 17583 [41]. Rectal temperatures and clinical signs of disease were recorded daily. Total and differential WBC and platelet counts were performed. Histologic examination and immunohistochemical analysis were conducted to detect lesions and distribution of viral antigens, respectively. RESULTS: Calves inoculated with BVDV 23025 or 17583 developed more severe clinical signs of disease (fever and diarrhea), more severe lymphopenia, and more severe lesions (alimentary epithelial necrosis, lymphoid depletion, and BVDV antigen deposition in lymphatic tissues), compared with calves inoculated with BVDV 713, 5521, or 17011. CONCLUSIONS AND CLINICAL RELEVANCE: Relative severity of experimentally induced infections corresponded to severity of clinical signs of naturally occurring infections with respective BVDV isolates.     

Fusobacterium equinum possesses a leukotoxin gene and exhibits leukotoxin activity

Fusobacterium equinum, a gram negative, rod-shaped and an obligate anaerobic bacterium is a newly described species. The organism is associated with necrotic infections of the respiratory tract in horses that include necrotizing pneumonia, pleuritis and paraoral infections. The species is closely related to F. necrophorum that causes liver abscesses in cattle and sheep, calf-diphtheria in cattle, and foot-rot in sheep and cattle. Leukotoxin, an exotoxin, is an important virulence factor in bovine strains of F. necrophorum. Our objective was to examine strains (n=10) of F. equinum for leukotoxin (lktA) gene and its toxic effects on equine leukocytes. Southern hybridization and partial DNA sequencing revealed that all the 10 strains had the lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The secreted leukotoxin was detected in the culture supernatant and its biological activity was determined by viability assays with equine polymorphonuclear cells (PMNs) using flow cytometry. While culture supernatants of four strains (E1, E7, E9, and E10) were highly toxic to equine PMNs; strain E5 was moderately toxic and the remaining strains (E2, E3, E4, E6, and E8) were only mildly toxic. Our data indicated that F. equinum isolates had lktA gene and its product was toxic to equine leukocytes. Therefore, leukotoxin may be an important virulence factor in F. equinum infections.     

The two major subspecies of Fusobacterium necrophorum have distinct leukotoxin operon promoter regions

Fusobacterium necrophorum, a gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two types of F. necrophorum, subspecies necrophorum (biotype A) and funduliforme (biotype B), have been recognized, which differ morphologically, biochemically and biologically. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis) such as bovine hepatic abscesses and ruminant foot abscesses. Subspecies necrophorum strains are considered to be more virulent for cattle and have been shown to produce greater amounts of leukotoxin than subspecies funduliforme strains. The leukotoxin operon of F. necrophorum consists of three genes (lktBAC) of which the leukotoxin structural gene (lktA) is the second gene in the operon. In this study, the promoter regions of the leukotoxin operons from the two subspecies were identified and their nucleotide sequence compared. The promoter regions were found to differ in sequence, in length of the sequence between the upstream determinant (oppF) and the first gene of the leukotoxin operon (lktB), and in promoter strength as assayed in Escherichia coli host cells.     

Establishment of an IL-2-dependent bovine T cell line and an assay of lymphocyte response inhibition in leukemic bovine serum.

The mechanism of immunosuppression induced by leukemic bovine serum was investigated with respect to lymphokine reactions using an interleukin 2 (IL-2)-dependent bovine T cell line generated from bovine peripheral blood lymphocytes (PBLs). The suppression of concanavalin A (con A)-induced PBL blastogenesis was observed at a high rate in leukemic cattle sera. The growth of IL-2-dependent bovine T cells and IL-2 production from con A-induced bovine PBLs were also inhibited by these sera, and particularly, the latter was correlated significantly to the degree of lymphocyte blastogenesis by the mitogen. Therefore, the lesser sensitivity of lymphocytes to IL-2 and the reduced IL-2 production by activated lymphocytes seem to play a role in suppressing the lymphocyte reaction.     

Minimum inhibitory concentrations for selected antimicrobial agents against Fusobacterium necrophorum isolated from hepatic abscesses in cattle and sheep

Minimum inhibitory concentrations for 35 antimicrobial agents against 100 Fusobacterium necrophorum isolates from hepatic abscesses in sheep and cattle were determined. Twelve of the thirteen β-lactam antibiotics tested inhibited growth of 100% of strains tested. Of the remaining antimicrobial agents, extensive susceptibility was found for: spiramycin, josamycin, lincomycin, tylosin, oxytetracycline, chlortetracycline, rufloxacin, metronidazole, co-trimoxazole, sulfadimethoxine, virginiamycin and fosfomycin.     

Fusobacterium necrophorum and Bacteroides melaninogenicus as etiologic agents of foot rot in cattle.

Fusobacterium necrophorum (Sphaerophorus necrophorus) and Bacteroides melaninogenicus were the predominant bacteria isolated from biopsy specimens of lesions in cattle affected with foot rot. Mixed inoculums of the 2 bacteria, applied to the scarified interdigital skin or inoculated intradermally into the interdigital skin of test cattle, induced typical lesions of foot rot. Both bacteria were reisolated in large numbers from the induced lesions.     

Experimentally induced Cooperia oncophora infection in calves: lymphocyte blastogenic and delayed hypersensitivity responses

Lymphocytic responses in peripheral blood and visceral lymph to Cooperia oncophora antigen and skin tests were determined in 35 Holstein male calves that were inoculated orally with single or multiple doses of C oncophora infective larvae. Several calves were vaccinated or given immune serum before larvae were inoculated. Antigen-specific in vitro blastogenesis of blood and lymph lymphocytes and delayed-type hypersensitivity reactions were observed in several inoculated, vaccinated, and/or passively immunized calves. Most calves that had delayed skin reactions also had in vitro lymphocyte responses to C oncophora antigen. The lymphocyte and skin responses were inconsistent and variable in time of onset--the earliest lymphocyte response occurring 7 days after calves were inoculated. A cellular immune response was induced by both dermal vaccination and oral inoculation; however, passive immunization by IV administration of immune serum simultaneously with inoculation did not have an apparent effect on the cellular response, as measured by the lymphocyte blastogenesis test or dermal testing. Although cellular immune responses were observed in several calves infected with C oncophora, there was no apparent relationship between the specific responses and number of nematodes establishing infection in calves after either single- or multiple-dose oral inoculations.     

Acute phase response in cattle infected with Anaplasma marginale

This study was undertaken to evaluate the acute phase responses via the assessment of the concentration of serum sialic acids (total, lipid bound and protein bound), inflammatory mediators (IFN-γ and TNF-α) and acute phase proteins (Hp and SAA) in 20 adult crossbred cattle naturally infected by Anaplasma marginale. The infected animals were divided into 2 subgroups on the basis of parasitemia rate (<20% and >20%). Also, as a control group, 10 clinically healthy cattle from the same farms were sampled. Our data revealed significant decreases in red blood cell count (RBC), hematocrite (PCV) and hemoglobine (Hb) in infected cattle compared to healthy ones. Conversely, the concentrations of Hp, SAA, ceruloplasmin, fibrinogen, serum sialic acids and the circulatory IFN-γ and TNF-α were increased in the diseased cattle (P<0.05). In addition, it was evident that the progression of parasitemia in infected cattle did not induce any significant alterations in the hematological indices (RBCs, PCV and Hb) and the concentrations of Hp, SAA, ceruloplasmin and fibrinogen. SAA was the most sensitive factor to change in the diseased cattle. Therefore, increase in SAA concentration may be a good indicator of inflammatory process in cattle naturally infected with Anaplasma marginale.     

Cell-mediated and humoral immune responses of cattle to Brucella abortus, Mycobacterium bovis, and tetanus toxoid: evaluation of immunization and assay techniques.

A concentration of 2.5 X 10(-5) M 2-mercaptoethanol (2-ME) added to the medium in lymphocyte blastogenesis assays increased both the uptake of [3H]thymidine in unstimulated lymphocyte cultures and the probability of detecting antigen-sensitized cattle. The use of 2-ME did not cause lymphocytes from unsensitized cattle to react positively in blastogenesis assays. A crude brucella lysate prepared from Brucella abortus strain 19 was compared with a well-characterized brucella protein allergen prepared from B melitensis and was found to be equally suitable for use in blastogenesis assays. Cell-mediated immunity was produced most effectively in 4-month-old calves by tetanus toxoid, then by Mycobacterium bovis, and least effectively by B abortus.     

Lesions and localization of viral antigen in tissues of cattle with experimentally induced or naturally acquired mucosal disease, or with naturally acquired chronic bovine viral diarrhea.

Tissues from cattle that died of experimentally induced mucosal disease (n = 3), naturally acquired mucosal disease (n = 6), or naturally acquired chronic bovine viral diarrhea (n = 4) were examined. Consistent findings were lymphocytic depletion of lymphoid tissues, degeneration of myenteric ganglion cells, and mild adrenalitis. Intracytoplasmic viral antigen was detected in myenteric ganglia and in endocrine glandular cells. Noncytopathic virus was isolated from all cattle, and cytopathic virus was isolated from 12 of 13 cattle.