Necrotizing granulomatous hepatitis in slaughtered broilers.

The present study describes a subclinical necrotizing granulomatous hepatitis in normal broilers routinely slaughtered in a medium-sized (72,000 birds per day) abattoir in the Netherlands. An exploratory investigation was scheduled on line during 20-min periods for 82 flocks (3000 birds examined per period). Liver and duodenum samples were collected for histopathology from 365 birds with liver pathology. Bacteriology was performed from 240 livers with lesions and 80 control livers. In addition to the hepatic pathology, other gross lesions of the carcasses, such as footpad dermatitis and broken legs/wings, were noted. The average prevalence for gross liver lesions was 0.16% (ranging from 0% to 0.63% per flock); 89.59% of the livers were enlarged, had a firm consistency, and revealed multifocal necrotic spots. Microscopically, 51.66% showed a granulomatous reaction in addition to the necrosis. There was no consistent anaerobic or aerobic bacterial growth in comparison to normal livers. A large proportion of the livers revealed growth of Escherichia coli, Bacteroides spp., Lactobacillus spp., Staphylococcus spp., and Streptococcus spp., and this was often with more than one type of bacterial colony. The duodenum mucosa grossly showed some redness with a mucous mass on its surface. Microscopically (n = 176) in 5.70% there were no changes in anatomy and cellular activities; 64.20% had a mildly increased number of lymphoid cells and heterophils in the lamina propria and between villus epithelial cells. The remaining 30.10% had moderate degenerative changes of villus epithelium with a mixed cellular infiltration in the lamina propria; 23.29% of the duodenum samples contained coccidia (infestation stage: mild to moderate). Signs of overgrowth with Clostridium spp. were not observed. There was a small, but significant correlation (rs = 0.30; P = 0.006) between prevalence of liver pathology and footpad dermatitis.

Pig acute-phase protein levels after stress induced by changes in the pattern of food administration.

A total of 240 pigs, 74 days old, half boars and half females, were included in a trial designed to assess the effect of the stress caused by changes in the pattern of food administration on the concentration of acute phase proteins (APP) and productive performance parameters. Half of the animals (pigs fed ad libitum, AL group) had free access to feed, while the rest were fed following a disorderly pattern (DIS group), in which animals had alternating periods of free access to feed and periods of no feeding, when food was removed from the feeder. The periods of free access to feed (two daily periods of 2-h duration) were randomly assigned, and varied from day to day. Total feed supplied per day was identical in both groups, and exceeded the minimal amount required for animals of these ages. Pen feed intake, individual body weights and the main positive pig APP pig major acute phase protein (Pig-MAP), haptoglobin, serum amyloid A (SAA), C-reactive protein (CRP), and the negative APP apolipoprotein A-I (ApoA-I) and transtherytin were determined every 2 weeks during the period 76 to 116 days of age. Animals fed ad libitum had better average daily gain (ADG) than DIS animals in the whole experimental period (P < 0.01) but the differences in ADG were only produced in the two first experimental sub-periods (60 to 74 and 74 to 116 days of age), suggesting that the stress diminished when the animals get used to the DIS feeding. Interestingly differences in ADG between DIS and AL pigs were due to males, whereas no differences were observed between females. The same differences observed for ADG were found for APP. DIS males had higher Pig-MAP concentration than AL males at 74 and 116 days of age, lower ApoA-I concentration at 74 days of age and higher haptoglobin and CRP concentration at 116 days of age (P < 0.05). The results obtained in this trial show an inverse relationship between weight gain and APP levels, and suggest that APP may be biomarkers for the evaluation of distress and welfare in pigs.

Avian hepatic granuloma. A review.

Hepatic granuloma is a chronic inflammatory disease characterized by a granulomatous reaction with accumulation of macrophages and/or epithelioid cells, which may fuse to form multinucleated giant cells. The hepatic granulomas typically have a surrounding rim of lymphocytes and fibrous tissues. The etiology of some hepatic granulomas in birds is well known. It could be due to viral, bacterial, fungal, protozoal, or helminthic infection. The presence of these pathogens in the liver is usually through systemic infections that might preferentially colonize the liver or be opportunistic invaders. Persistence of these pathogens infecting the liver can lead to granulomatous inflammation with different gross lesions and histopathologic patterns depending on the causative agent. This review describes the etiology, clinical signs, pathological changes, and diagnosis in a wide variety of diseases associated with hepatic granulomas in birds in which the detection of granulomatous inflammation is an aid in the differential diagnosis.

An overview of swine influenza.

Swine influenza is a highly infectious viral disease of pigs, causing considerable economic impact. The causative agent is known as a type A orthomyxovirus with a segmented RNA genome. Influenza type A virus is a highly contagious pathogen among a limited number of birds and mammals. The objective of this review is to summarize the current knowledge in swine influenza infection in pigs with emphasizing on epidemiology, pathogenesis, diagnostic techniques and control measures.

Relationship of plasma nonesterified fatty acids and walking activity in postpartum dairy cows.

To survive and produce milk, postpartum dairy cows use their reserves through lipolysis. If the negative energy balance is severe, nonesterified fatty acids (NEFA) are formed that can impair several physiological processes. A pilot study suggested that increased walking activity after calving may be related to a reduced serum concentration of NEFA. The objective of this study was to determine the relationship between plasma concentrations of NEFA and walking activity in dairy cattle during the postpartum period. Data were collected from 33 multiparous Holstein-Friesian dairy cows. Walking activities were quantified using pedometry, and blood samples were collected for determination of NEFA. Results of this study indicated that a negative relationship existed between walking activity and plasma NEFA concentrations in postpartum dairy cows.

The porcine acute phase protein response to acute clinical and subclinical experimental infection with Streptococcus suis.

The pig acute phase protein (APP) response to experimental Streptococcus suis (S. suis) infection was mapped by the measurement of the positive APPs C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp) and major acute phase protein (pig-MAP) and the negative APPs albumin and apolipoprotein (Apo) A-I. The aim was to elucidate the differences in the acute phase behaviour of the individual APPs during a typical bacterial septicaemic infection. Pigs were inoculated subcutaneously with live S. suis serotype 2 and blood was sampled before and on various days post inoculation (p.i.), until the pigs were killed and autopsied on day 14 p.i. Clinical signs (fever and lameness) were observed in four of the five inoculated pigs from day 2 p.i., and these pigs also had arthritic lesions at autopsy. CRP and SAA showed fast increases in serum concentrations, CRP being elevated from days 1 to 12 p.i. and peaking at 10 times the day 0-levels on day 1 p.i. SAA rose quickly to peak levels of 30-40 times the day 0-level on days 1-2 and returned to pre-inoculation level on day 5 p.i. Hp and pig-MAP showed slightly slower responses, both peaking around 5 days p.i. Hp was increased throughout the experiment with maximum levels around 10 times the day 0-levels, and pig-MAP was elevated on days 1-12 p.i. with peak levels of around seven times the day 0-levels. Apo A-I was decreased from days 1 to 8 and showed minimum levels of about 40% of day 0-levels around 1-2 days p.i. No clear pattern of changes in albumin levels could be identified. One pig, showing clinical signs on day 2 only, also showed an APP response, although of a relatively short duration, whereas three pigs presenting clinical signs for several days had a more protracted acute phase response. Remarkably, the one pig showing no clinical signs and no arthritic lesions showed an APP response comparable to that of the other, clinically affected pigs. Thus, both acute clinical and subclinical S. suis infection could be revealed by the measurement of one or more of the APPs CRP, SAA, Hp, pig-MAP and Apo A-I. The combined measurement of two or three APPs, including proteins with slow and fast kinetics, should be used to achieve the highest sensitivity for the detection of ongoing S. suis infection during a prolonged time period. A diagnostic tool based on such APP-measurements could considerably improve strategic control procedures for this important infection.

Dry gangrene of the extremities in calves associated with Salmonella dublin infection; a possible immune-mediated reaction.

Dry gangrene of the extremities in calves is a circulatory error that may occur after infection with Salmonella dublin. This report describes an examination of three affected, 12 in-contact and five control calves, a main objective being to investigate the possible role of cold agglutination in pathogenesis. The lesions included dry gangrene of the hind legs, ears and tail. A cold agglutination test gave positive results in all animals examined except the controls. The three affected calves had high titres of S. dublin antibodies, as also did four of the in-contact animals. The results suggested a relationship between cold agglutination and the occurrence of the disease.

Monitoring health by values of acute phase proteins.

A systemic acute phase reaction may develop during infection and inflammation, due to the action of peripherally liberated proinflammatory cytokines. Hepatic metabolism changes, and negative and positive acute phase proteins (APPs) can be measured in the blood: the APPs therefore represent appropriate analytes to assess health. While they are non-specific markers, their levels change with biological effects and this can be used to assess nutritional deficits and reactive processes, especially when positive and negative acute phase variables are combined in an index. Unfortunately, at present, no comprehensive, easy-to-use and cheap system is available to assess various acute phase proteins in serum or blood samples. Protein micro-array technology may satisfy this need; it will permit simultaneous analysis of numerous analytes in the same small volume sample and enable integration of information derived from systemic reactivity and nutrition with disease-specific variables. Applying such technology may help to address health problems in many countries.

Cognitive disturbances in old dogs suffering from the canine counterpart of Alzheimer's disease.

In geriatric dogs, Alzheimer-like behavior is frequently observed. This behavior has been classified by several authors using questionnaires and a correlation has been described between cognitive dysfunctions and Alzheimer-like pathology. In the present study, cognitive performance was correlated with brain pathology for 30 dogs of varying ages. Within these animals, two age-matched groups of old dogs with and without behavioral changes were compared. The behavioral changes were analyzed and scored with questionnaires and necropsy was performed to rule out any other cause for changed behavior. Measurements, (immuno)-histochemical staining and fluorescence microscopy were used to detect cortex atrophy, amyloid, rest-products of oxidative damage, demyelination and accumulations of macrophages in the brains of these dogs. Spearman rank correlation coefficients (r) were calculated and adjusted according to Bonferonni. In the whole group (young to very old dogs), the age of the animal showed a significant correlation with various behavioral changes (r = 0.7 to 0.9, P < 0.01). The dementia score correlated significantly (r = 0.6 to 0.8, P < 0.01) with all the brain lesions studied, except one, i.e. demyelination (r = -0.4, P > 0.05). These results suggest that a questionnaire can be used to diagnose Alzheimer-like changes in canine practice. Oxidative damage on a cellular and a nuclear level plays an important role in behavior changes.

Acute phase reaction and acute phase proteins.

A review of the systemic acute phase reaction with major cytokines involved, and the hepatic metabolic changes, negative and positive acute phase proteins (APPs) with function and associated pathology is given. It appears that APPs represent appropriate analytes for assessment of animal health. Whereas they represent non-specific markers as biological effect reactants, they can be used for assessing nutritional deficits and reactive processes, especially when positive and negative acute phase variables are combined in an index. When such acute phase index is applied to separate healthy animals from animals with some disease, much better results are obtained than with single analytes and statistically acceptable results for culling individual animals may be reached. Unfortunately at present no cheap, comprehensive and easy to use system is available for assessing various acute phase proteins in serum or blood samples at the same time. Protein microarray or fluid phase microchip technology may satisfy this need; and permit simultaneous analysis of numerous analytes in the same small volume sample and enable integration of information derived from systemic reactivity and nutrition with disease specific variables. Applying such technology may help to solve health problems in various countries not only in animal husbandry but also in human populations.

Acute phase reactants, challenge in the near future of animal production and veterinary medicine.

The future of acute phase proteins (APPs) in science is discussed in this paper. Many functions and associated pathological processes of APPs are unknown. Extrahepatic formation in local tissues needs attention. Local serum amyloid A (SAA) formation may be involved in deposition of AA-amyloid induced by conformational change of SAA resulting in amyloid formation, having tremendous food safety implications. Amyloidogenesis is enhanced in mouse fed beta pleated sheet-rich proteins. The local amyloid in joints of chicken and mammary corpora amylacea is discussed. Differences in glycosylation of glycoproteins among the APPs, as has been shown for alpha1-acid glycoprotein, have to be considered. More knowledge on the reactivity patterns may lead to implication of APPs in the diagnostics and staging of a disease. Calculation of an index from values of several acute phase variables increases the power of APPs in monitoring unhealthy individuals in animal populations. Vaccinations, just as infections in eliciting acute phase response seem to limit the profitability of vaccines because acute phase reactions are contra-productive in view of muscle anabolism. Interest is focused on amino acid patterns and vitamins in view of dietary nutrition effect on sick and convalescing animals. When inexpensive methodology such as liquid phase methods (nephelometry, turbidimetry) or protein array technology for rapid APP measurement is available, APPs have a future in routine diagnostics. Specific groups of patients may be screened or populations monitored by using APP.

Systemic AA-amyloidosis in a European wild boar (Sus scrofa) suffering from generalized tuberculosis.

Summary An adult female wild boar (Sus scrofa) was found moribund in Cabaneros National Park (central Spain). The animal had a markedly emaciated carcass, with body weight of 25.9 kg. At necropsy, most of the parenchymatous organs had widespread variably sized granulomas. Generalized tuberculosis was confirmed by PCR detection of Mycobacterium bovis in the mandibular lymph node. Large amounts of a hyaline, pale eosinophilic material were observed in liver, kidney and intestine. Congo red staining and green birefringence identified amyloid, which was further classified as AA type based on immunohistochemical results. It is speculated that the abundant serum amyloid A derivatives deposited in the tissues as AA-amyloid may be associated with the generalized tuberculosis. This is the first report of amyloidosis in the European wild boar.

Kinetics of local and systemic isoforms of serum amyloid A in bovine mastitic milk.

The aim of the present study was to characterise the serum amyloid A (SAA) response to intramammary inoculation of Escherichia coli and to examine the distribution of hepatically and extrahepatically produced SAA isoforms in plasma and milk from cows with mastitis. Milk and plasma SAA concentrations were determined before and after experimental induction of E. coli mastitis in six dairy cows. The milk SAA response was characterised by low or undetectable levels before inoculation, very rapid and large increases in concentration after inoculation, and rapid decline towards baseline levels after resolution of disease. In plasma from cows with experimentally induced E. coli mastitis, four hepatically derived SAA isoforms with apparent isoelectric point (pI) values of 5.8, 6.2, 6.8 and 7.4 were demonstrated by denaturing isoelectric focusing. In milk three highly alkaline isoforms with apparent pI values above 9.3 appeared 12 h post-inoculation. These isoforms were not present in any of the plasma samples, and it therefore seems likely that they were locally produced, tissue-specific isoforms. At 24-36 h post-inoculation one or more acidic isoforms corresponding to those found in plasma appeared in the milk samples. The isoforms demonstrated in plasma from cows with E. coli mastitis were also present in serum obtained from three cows with clinical Streptococcus uberis mastitis. In conclusion, experimentally induced E. coli mastitis is accompanied by a prominent SAA response. The results of the present study indicate that SAA accumulation in mastitic milk is the result of both local synthesis of SAA and of hepatically derived SAA gaining access to the milk due to increased permeability of the blood-milk barrier.

The acute phase response of haptoglobin and serum amyloid A (SAA) in cattle undergoing experimental infection with bovine respiratory syncytial virus.

The ability of a pure virus infection to induce an acute phase protein response is of interest as viral infections are normally considered to be less efficient in inducing an acute phase protein response than bacterial infections. This was studied in a bovine model for infection with bovine respiratory syncytial virus (BRSV), analysing the induction of the two most dominant bovine acute phase proteins haptoglobin and serum amyloid A (SAA). Strong and reproducible acute phase responses were detected for both proteins, peaking at around 7-8 days after inoculation of BRSV, while no response was seen in mock-inoculated control animals. The serum concentrations reached for SAA and haptoglobin during the BRSV-induced acute phase response were generally the same or higher than previously reported for bacterial infections in calves. The magnitude and the duration of the haptoglobin response was found to correlate well with the severity of clinical signs (fever) and with the extent of lung consolidation while SAA responded most rapidly to infection.

Serum amyloid A and transferrin in chicken. A preliminary investigation of using acute-phase variables to assess diseases in chickens.

The concentrations of serum amyloid A protein (SAA) and transferrin in blood samples from broilers in various stages of natural Staphylococcus aureus infection, from healthy counterparts, and from turpentine- or saline-injected pullets were measured using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and densitometry. SAA was not detected in healthy chickens but was detected in turpentine-injected pullets and in S. aureus-infected broilers. Relative percentages of transferrin in turpentine- and saline-injected pullets were not different. Broilers with a S. aureus infection had a two-fold higher transferrin level than did their unaffected counterparts. There was also a difference between the transferrin levels of healthy broilers and healthy pullets, which indicates that other variables, such as breed or breeding conditions, may influence the transferrin level. This preliminary study showed that SAA is an acute-phase protein and a more reliable variable for diagnosing lesions in chickens than transferrin.

Lack of effect of extracellular matrix or 3T3 feeder layer on the maintenance of differentiation or survival time of cultured rat hepatocytes.

Cultured rat hepatocytes are often used in in vitro studies. In this culture system the lack of the normal environment to which the cells would be exposed in vivo contributes to a change in differentiation. To study this differentiation problem we compared the effects of culturing primary hepatocytes on an extracellular matrix (ECM) isolated from normal rat livers and on a 3T3 (mouse fibroblast) feeder layer. The use of the two coatings did not result in changes in survival time, as measured by leakage of lactate dehydrogenase, differentiation (activities of pyruvate kinase and gamma-glutamyltransferase) or specific functions (albumin production and concentration of cytochrome P-450) in comparison with cells cultured on plastic. Since no positive effects of the coatings were found it is proposed that more attention should be paid to methods of isolating and culturing hepatocytes, rather than to obtaining conditions for the hepatocytes that are completely comparable with the in vivo situation.

Phenobarbital pretreatment in vivo and in vitro and the effect of hepatotoxicity of d-galactosamine in rat hepatocytes in culture.

Galactosamine (GalN) is a known hepatotoxic compound, acting by depletion of uracil nucleotides. The relation between an active cytochrome P-450 system (CYP) and the hepatotoxicity of GalN was studied in rat hepatocytes that were pretreated with phenobarbital (PB) in vivo or in vitro. A 24-hr in vitro pretreatment of cultured hepatocytes with PB resulted in a significant decrease in GalN toxicity as measured by lactate dehydrogenase (LDH) leakage. Furthermore, GalN treatment resulted in an increase in the activity of the PB-induced forms of CYP (namely CYP 2B1/2) as measured by 7-pentoxyresorufin O-depentylase (PROD) activity. This increase was not found after GalN treatment of microsomes. GalN had no effect on the concentration of the apoenzymes. GalN administration to hepatocytes of in vivo PB-pretreated rats resulted in a similar effect of GalN on the activity of the CYP enzymes but PB in vivo had no effect on GalN toxicity. These results suggest that GalN treatment may result in a significant increase in the specific activity of CYP 2B1/2 enzymes (PROD), without an obvious increase in the amount of PB-induced apoenzymes. This phenomenon was measurable only in intact cells. No direct relation is assumed between the activity of the CYP apoenzymes and the decrease in GalN toxicity after PB treatment. The toxicity of Galn was inhibited by PB treatment in vitro.

Copper and zinc toxicity in two rat hepatoma cell lines varying in differentiation.

1. The differentiation status of cells is considered to represent an important factor in determining the effects of toxic components. 2. Two rat hepatoma cell lines, MH1C1 and HTC, were used to study differences in the sensitivity to two toxic metals: copper and zinc. 3. The differentiation of the cell lines was characterized using light microscopy, growth pattern, gamma glutamyltransferase (GGT) activity and albumin production as parameters. 4. The MH1C1 cell line was described to be more differentiated. 5. Albumin production in the MH1C1 cells was significantly higher than in the HTC cells whereas the GGT activity was only slightly different. 6. Toxicity of Cu and Zn was compared. Zn appeared to be more toxic to the cells than Cu, when leakage of lactate dehydrogenase and potassium were measured, whereas both metals were equally toxic when expressed as DNA remaining after 24 hr. 7. The MH1C1 cell line appeared to be more sensitive to Cu and Zn than the HTC. 8. The two metals appeared to have different targets in the cell; Cu may affect the nucleus and Zn the cell membrane.

Effects of cytokines, bacterial endotoxin and hydrocortisone on primary hepatocyte cultures of rat and hamster.

In vivo an acute phase reaction is characterized by changes in hepatic protein metabolism and modification of liver drug enzymes (cytochrome P-450). In vitro, mediators of an acute phase reaction have been reported to have a similar effect. In the present study, the effects of bacterial endotoxin (lipopolysaccharide, LPS) and cytokines in hepatocyte cultures of rat and hamster were examined. In both species, a significant increase in secretion of specific acute phase protein was measured after 48 hr of exposure to IL-6, TNF-alpha or LPS. LPS and TNF-alpha increased the secretion of serum amyloid A (SAA) in hamster hepatocytes. LPS and IL-6 (but only in the presence of hydrocortisone) increased the secretion of alpha(2)-macroglobulin (alpha(2)M) in rat hepatocytes. An effect on the total concentration of cytochrome P-450 was not found in either species after exposure to glucocorticoids or to LPS and cytokines, although the latter compounds caused a marked increase in secretion of acute phase proteins.