Mechanism of thrombocytopenia in African swine fever

Pigs were inoculated with an African swine fever (ASF) isolate of moderate virulence, and the changes in the number of circulating blood platelets during infection were correlated with the appearance of antiviral antibody and fluctuations in total plasma hemolytic complement concentrations. Thrombocytopenia was detected by postinoculation days (PID) 7 and 8, and antiviral antibody was detected by PID 7, using an indirect immunofluorescence technique. The total hemolytic complement concentration was moderately and transiently decreased from PID 5 to 9, but was consistently low from PID 18 to 26. Pigs inoculated with an ASF virus isolate of greater virulence had a decrease in platelet counts on PID 6 and 7, and the total plasma hemolytic complement levels decreased in all pigs by PID 6 to 7. Antibody to ASF virus was not detected in pigs inoculated with the more virulent isolate. Pigs sensitized to ASF viral antigen with an inactivated-virus vaccine or by previous infection with ASF were challenge exposed. Sensitized pigs became clinically ill and thrombocytopenic by 24 to 72 hours earlier than did inoculated, nonsensitized pigs. Vaccinated pigs inoculated with homologous virus had lower blood virus concentrations than did nonvaccinated pigs. African swine fever virus-sensitized pigs inoculated with heterologous virus had a higher fatality rate than did nonsensitized pigs, and the pigs died peracutely, with only a few gross lesions in evidence. In vitro experiments demonstrated that ASF virus antigen induced platelet aggregation in platelet-rich plasma from recovered, nonviremic pigs. Viral antigen, antibody, or complement was not demonstrable on the surface of platelets from pigs inoculated with ASF virus isolate, by direct immunofluorescence testing.     

Acute phase protein response during subclinical infection of pigs with H1N1 swine influenza virus

In the present study acute phase proteins (APPs) responses in pigs after subclinical infection with H1N1 swine influenza virus (SwH1N1) were evaluated. Fourteen 5 weeks old, seronegative piglets, both sexes were used. Ten of them were infected intranasally with SwH1N1. C-reactive protein (CRP), haptoglobin (Hp), serum amyloid A (SAA) and pig major acute phase protein (Pig-MAP) concentrations in serum were measured using commercial ELISAs. No significant clinical signs were observed in any of the infected pigs, however, all infected animals developed specific antibodies against SwH1N1 and viral shedding was observed from 2 to 5dpi. Only concentrations of Hp and SAA were significantly induced after infection, with mean maximum levels from days 1 to 2 post infection (dpi). The concentrations of CRP and Pig-MAP remained generally unchanged, however in half of infected pigs the concentration of CRP tended to increase at 1dpi (but without statistical significance). The results of our study confirmed that monitoring of APPs may be useful for detection of subclinically infected pigs. The use of SAA or Hp and Pig-MAP may be a valuable in combination [i.e. Hp (increased concentration) and Pig-MAP (unchanged concentration)] to detect subclinically SIV infected pigs, or to identify pigs actually producing a large amount of virus. Additional studies need to be done in order to confirm these findings.     

Coagulation changes in African swine fever virus infection

Pigs were infected with highly virulent (Tengani '62), with moderately virulent (DR '79) African swine fever (ASF) virus, or with virulent hog cholera (HC) virus. Changes in platelet counts, selected coagulation assays and concentrations of factor VIII-related antigen (VIIIR:Ag) were monitored. Permeability of aortic endothelium was studied after the injection of Evan's blue dye on various days after infection with DR '79 ASF virus. Virulent ASF virus caused prolongation of the activated partial thromboplastin time (APTT), 1-stage prothrombin time, and thrombin clotting time as early as postinoculation day (PID) 4. These changes became progressively more severe until death. Both virulent HC and DR'79 viruses induced an increase APPT and thrombin clotting time at PID 3 to 4, only occasionally did the prothrombin time increased significantly (P less than 0.01). The APPT began to decrease on PID 7 and 8, but only DR'79-infected pigs lived long enough to regain a normal APTT. Infection by ASF viruses caused acute thrombocytopenia after PID 6 and platelet counts of HC virus-infected pigs decreased progressively from the onset of fever to levels of 1 to 2 X 10(5)/mm3 at PID 6 to 7. All ASF virus-infected pigs had an increase in VIIIR:Ag beginning at PID 3, with maximum increases at PID 6 to 7. Hog cholera virus infection did not cause consistent changes in levels of VIIIR:Ag. Pigs infected with DR'79 virus did not have increased vascular permeability to Evan's blue dye during infection; however, there was markedly decreased staining of the aorta after pigs became thrombocytopenic.     

The role of acute phase proteins in diagnosis and management of steroid-responsive meningitis arteritis in dogs

Acute phase proteins (APPs) have become an important tool in the diagnosis, management and prognosis of inflammatory diseases in humans and are developing a similar utility in domestic species. Steroid-responsive meningitis arteritis (SRMA) is a well-recognised inflammatory disease of the dog, the diagnosis of which remains unsatisfactory based on clinical criteria and non-specific laboratory investigations. In this prospective pilot study the authors examined the acute phase response throughout the course of SRMA in serum and cerebrospinal fluid (CSF) by evaluating three key stages in disease management: presentation, treatment response and putative relapse.Serum APPs were found to be of value in supporting the diagnosis of SRMA and monitoring its treatment. C-reactive protein (CRP), serum amyloid-A (SAA), alpha-1-acid glycoprotein (AGP) and haptoglobin (Hp) all exhibited an increase above our laboratory reference range in nine patients at initial presentation. During treatment APPs decreased significantly compared to presentation except Hp which increased (Wilcoxon–Signed–Rank-test: CRP, SAA and AGP P < 0.05). Serum CRP and SAA were also found to be of clinical value in the identification of putative relapse (seven cases), particularly in the light of unperturbed CSF parameters where APP concentrations were elevated. CSF APPs were found to be less reliable markers in the management of this disease. The results indicate that SRMA causes a significant APP response in dogs, which although not disease specific, is of value in supporting the diagnosis of SRMA.     

A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples

The development and standardisation of a novel, highly sensitive and specific one-step hot start multiplex RT-PCR assay is presented for the simultaneous and differential diagnosis of African swine fever (ASF) and Classical swine fever (CSF). The method uses two primer sets, each one specific for the corresponding virus, amplifying DNA fragments different in length, allowing a gel-based differential detection of the PCR products. Universal detection of ASF and CSF virus strains was achieved through selection of primers in conserved viral genome regions. The detection range was confirmed by analysis of a large collection of isolates of the two viruses. The high specificity of the assay was proven by testing related viruses, uninfected cell line cultures and healthy pig tissues. Additional confirmatory tests of the ASF and CSF virus amplicon specificity, based on restriction endonuclease analysis with BsmA I or Ban II, respectively, are also described. The analysis of whole blood and serum samples from experimentally infected animals proved the usefulness of the method for an early diagnosis of both diseases, even before the appearance of the first clinical signs. A study of 150 positive field samples from several ASF and CSF outbreaks showed the suitability of this method for a rapid (less than five hours), sensitive and specific differential diagnosis in clinical samples. In addition, a highly sensitive and specific uniplex RT-PCR for CSFV was also developed and standardised as a powerful tool for fast and early diagnosis of the disease.     

Porcine acute phase protein concentrations in different diseases in field conditions

Five acute phase proteins (APPs) [C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), pig-MAP and albumin] were measured in pigs with naturally occurring infections by porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky's disease virus (ADV), porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae, and in animals with tail and ear bites, arthritis and other acute inflammatory processes. Healthy specific pathogen-free (SPF) pigs were used as controls. In PRRSV-infected pigs, all APPs with the exception of pig-MAP exhibited significant changes compared with controls. In animals affected with ADV only Hp presented changes of statistical significance, whereas pigs with PCV2 showed marked modifications in all APPs tested. Animals affected with Mycoplasmosis showed concentrations of all positive APPs significantly above levels obtained in SPF pigs, though albumin concentrations did not differ from controls. Finally, all APPs studied showed substantial changes in pigs with acute inflammation. The results indicated that an acute phase response was developed in the different diseases studied, this response being higher in animals with clinical signs and concurrent bacterial processes. Haptoglobin would be the APP that better reflects pathological states; however, to get more complete and valuable information it might be advisable to perform APPs profiles including another APP, such as CRP or SAA.     

Inhibition of African swine fever infection in the presence of immune sera in vivo and in vitro

In assay cultures, sera from African swine fever convalescent pigs inhibited infection by homologous African swine fever virus. The infection-inhibition capacity did not correspond with the virus-neutralizing capacity. The serum did not prevent infection by heterologous virulent viruses. Sera from pigs challenge inoculated with the homologous virulent virus and later with a heterologous virulent virus inhibited the infection by different heterologous virulent viruses. These sera did not interfere with the infection by pseudorabies virus. The specificity of the reaction indicated that the infection inhibition was caused by antibody.     

Acute phase proteins in cattle and swine: A review

The major acute phase proteins (APPs) in cattle are haptoglobin (Hp) and serum amyloid A (SAA), and in swine, are Hp, SAA, C-reactive protein (CRP), and Pig major acute phase protein (Pig-MAP). Many methodologic assays are presently available to measure these parameters, which are still being improved to increase their specificity, sensitivity, user-friendliness, and economic availability. In cattle, the main applications are the diagnosis and monitoring of frequent diseases such as mastitis and metritis in dairy cows and respiratory problems in young calves. In pigs, APPs are useful in the control of bacterial and viral infections, and they may be used at the slaughterhouse to monitor subclinical pathologies and improve food safety. The utility of APP in animal production must not be forgotten; optimization of protocols to improve performance, welfare, and nutrition may benefit from the use of APPs. Other sample types besides serum or plasma have potential uses; APP determination in milk is a powerful tool in the control of mastitis, saliva is a non-invasive sample type, and meat juice is easily obtained at the slaughterhouse. Increasing our knowledge of reference intervals and the influence of variables such as age, breed, sex, and the season is important. Finally, worldwide harmonization and standardization of analytical procedures will help to expand the use of APPs.     

非洲猪瘟研究进展及防控措施

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒科、非洲猪瘟病毒属的唯一成员非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种高致死率、高抵抗力、严格接触性的病毒性疾病,对所有年龄段的家猪和野猪均具有感染性。我国非洲猪瘟传入疫情的出现,对养猪生产构成严重威胁,必将冲击生猪产业的健康稳定发展。本文介绍ASF的病原、流行病学特点,并总结防控措施,以期为ASF的进一步防控提供参考。     

Preliminary studies of serum acute-phase protein concentrations in hematologic and neoplastic diseases of the dog

Serum concentrations of acute-phase proteins (APPs): haptoglobin (Hp), ceruloplasmin (Cp), serum amyloid A (SAA), and C-reactive protein (CRP) were determined in healthy dogs (n = 15) and dogs with different diseases grouped as acute inflammation (I, n = 12), hematologic neoplasias (HT, including leukemia and lymphoma, n = 16), nonhematologic neoplasias (NHT, including epithelial, mesenchymal, and mixed, n = 20), and autoimmune hemolytic anemia (AIHA, n = 8). SAA and CRP were analyzed using commercially available enzyme-linked immunosorbent assay (ELISA) kits, and Hp and Cp were measured using colorimetric methods, all previously validated for use in dogs. Increased concentrations of all APPs were observed in all groups of diseased dogs, but statistical significance only was observed with Hp (I, P < .001; HT, P < .05), Cp (I, P < .05; AIHA, P < .01), and CRP (I, P < .001; HT, P < .001; AIHA, CRP P < .05). High variability in individual APPs within each group of diseases was found with no significant differences between leukemia and lymphoma as well as among different types of neoplasia. The AIHA group had smaller increases in Hp, SAA, and CRP but higher concentrations of Cp. When follow-up of individual cases was possible, a decrease in APPs generally was found in cases with favorable outcome. The results of this study suggest that neoplasia and hematologic diseases such as AIHA should be considered as possible causes of mild increases in APPs in dogs. Measurement of APPs may be helpful to assess clinical evolution and monitor treatment of these processes.     

An immunohistochemical study of the tonsils in pigs with acute African swine fever virus infection

An immunohistochemical study of the tonsils was carried out to gain further insight in the pathogenesis of acute African swine fever (ASF). Twenty-one pigs were inoculated by intramuscular route with a highly virulent isolate of ASF virus and painlessly killed at 1-7dpi. Viral antigen was highly distributed in the tonsil from 3 to 4dpi and an increase in the number of monocyte-macrophages was very evident at the same days post inoculation. This phenomenon was observed together with an increase of the expression of proinflammatory cytokines (Tumour necrosis factor alpha and Interleukin-1 alpha) and the apoptosis of lymphocytes studied by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) technique and haemorrhages. With these results, we can conclude that the tonsil is suffering similar lesions than those observed in other lymphoid organs in acute African swine fever, even when the route of inoculation is the intramuscular and not oral-nasal.     

Detection of African swine fever viral antigens in paraffin-embedded tissues by use of immunohistologic methods and polyclonal antibodies.

Tissues obtained from pigs inoculated with African swine fever virus (ASFV), fixed by vascular perfusion using glutaraldehyde, and embedded in paraffin or araldite were used for an immunohistologic electron microscopic study. To detect ASFV antigens, 4 methods were used on paraffin sections with or without pretreatment of the tissues. Use of biotinylated anti-ASFV antiserum combined with avidin-biotin complex and peroxidase proved to be the most suitable method, and antigen was detected in tissues infected with 2 ASF viruses of different virulence. Use of the glutaraldehyde fixation method should ensure optimal morphologic (structural and ultrastructural) data while allowing an immunohistologic study, and add to knowledge of the pathogenesis of ASF.     

Evidence of indirect transmission of classical swine fever virus through contacts with people

A strict system for visiting experimentally inoculated and susceptible weaner pigs was used to examine the potential indirect transmission of classical swine fever (CSF) virus by people wearing contaminated boots, gloves and coveralls. The inoculated and susceptible pigs were housed in separate compartments, between which the airborne transmission of the virus was impossible. A worst-case scenario with an intensive visiting protocol and no form of disinfection or hygiene was established. Fifteen days after the pigs were inoculated, infection was detected in one contact pig, and it was concluded that under the conditions of the experiment CSF virus could be transmitted by contact with people.     

Experimental transmission of African swine fever virus by Ornithodoros savignyi (Audouin)

The 'sand tampan', Ornithodoros savignyi, is susceptible to oral infection with African swine fever (ASF) virus in the laboratory. Infected ticks can transmit the virus transstadially and are able to maintain it for at least 106 days. Transmission of ASF virus by infected ticks to healthy pigs was achieved on five separate occasions between 50 and 106 days after infection. Pigs infected in this way developed typical acute African swine fever. The distribution of O savignyi in Africa suggests that this tick could be a natural field vector of ASF.     

Steroid Responsive Meningitis-Arteritis: A Prospective Study of Potential Disease Markers, Prednisolone Treatment, and Long-Term Outcome in 20 Dogs (2006–2008)

Background: Previous multidrug studies have identified the value of prednisolone in treating steroid responsive meningitis-arteritis (SRMA) and the potential value of acute phase proteins (APPs) and immunoglobulin A (IgA) in diagnosis and monitoring.
Hypothesis: (1) Prednisolone monotherapy is a successful immunosuppressive modality in the treatment of SRMA; (2) protein markers are useful in identifying the potential for relapse.
Animals: Twenty client-owned dogs with SRMA presented to the University of Glasgow Small Animal Hospital between May 2006 and May 2008.
Methods: A prospective, observational study: CBC, biochemistry, and cerebrospinal fluid (CSF) analyses were performed. C-reactive protein (CRP), serum amyloid-A, α-1-acid glycoprotein, and haptoglobin (Hp) were assessed in the serum. IgA concentrations were determined in the serum and CSF.
Results: Clinical resolution of SRMA was achieved in all 20 dogs. Serum CRP concentration remained increased at remission in 16/20 dogs whereas CSF cytology was within normal limits in 20/20 dogs. Serum APPs decreased significantly on treatment ( P < .05) except Hp, which remained unaltered. Serum and CSF IgA concentrations remained increased for the duration of treatment.
Conclusions and Clinical Importance: The prednisolone regimen presented was successful in treating SRMA without the need for additional drugs. Serum APPs are of use in the diagnosis and management of SRMA, particularly in relation to identifying relapse. Serum and CSF IgA concentrations remain increased throughout disease, aiding in diagnosis but not contributing to the management of SRMA.     

Phylogenetic analysis of recent classical swine fever virus (CSFV) isolates from Assam, India

Classical swine fever (CSF), a highly contagious viral disease of pigs, is endemic in India. As there is no information concerning the accurate genetic typing of classical swine fever virus (CSFV) isolates in India, 16 CSF viruses isolated during 2005-2007 from domestic pigs in different districts of Assam were typed in 5′ UTR (150 nucleotides). To confirm the genetic typing results and to study the genetic variability, selected viruses were also analyzed in E2 (190 nt) and NS5B gene (409 nt) regions. Phylogenetic analysis revealed that all the 16 CSFV isolates analyzed belonged to group 1 and subgroup 1.1 in contrast to the situation in other Asian countries. Additionally, analysis in E2 and NS5B region placed the Indian isolates in a clearly separated clade within subgroup 1.1. The results suggest that subgroup 1.1 CSF viruses are currently circulating in India, which is important for epidemiology and control of CSF.     

Extraction of viral DNA from erythrocytes of swine with acute African swine fever

The preparation of wild-type African swine fever (ASF) virus DNA from small amounts of viremic blood from acutely febrile pigs is outlined. The extracted DNA is viral and not host-cell DNA, because of specific homology with cell culture grown and purified ASF virus and because no DNA bands are obtained with an equal amount of nonviremic pig blood. Thus, in the absence of suitable serologic methods for strain identification, it is now possible to catalogue wild-type isolates by characteristic DNA restriction patterns. The wild-type virus genome contains terminal single-stranded DNA cross-links and has the largest genome size (180 kilobase pairs) reported for the ASF virus. Experimental passage of the virus in contact-infected pigs and buffy coat cultures appears to confirm the stable nature of the ASF genome in the field.     

Diversity of African swine fever virus

An African swine fever virus is an heterogeneous population, consisting of clones having different biological characteristics in respect to hemadsorption, virulence, infectivity, plaque size, and antigenic determinants. The following observations were made: Nonhemadsorbing virus (NHV) have been segregated from field isolates from Haiti (HT-1) and a bone marrow- and buffy coat-passaged Portuguese isolate (L'60BM89BC1) and appear as a major, minor, or equal mixture with hemadsorbing viruses in the virus population. Biological characteristics of the virus inoculated into pigs often differed from viruses isolated later from the same pigs. Virulence and nonhemadsorbing characteristics of isolated clones were genetically stable. The lethal effect of 2 NHV clones of L'60BM89BC1 virus was dose-dependent; small doses of virus induced immunologic deaths or recoveries from the clinical disease in pigs, and large doses induced acute deaths. The NHV of Lisbon isolate of 1960 (L'60) and HT-1 isolate share the same antigenic determinants for inducing protection. Tengani isolate contained clones of distinctly different antigenic determinants, not shared by L'60 or HT-1 isolate that enabled it to overcome the protection induced by the other clones. Passaging of an African swine fever virus isolate in pigs or cell cultures may readily alter the proportions of the different clones in the population and thereby change its overall characteristics. A new virus population with atypical hemadsorption was found in HT-1 field isolate and L'60BM89BC1 virus.     

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.     

Analytical validation of commercially available methods for acute phase proteins quantification in pigs

The aim of this study was to validate commercially available methods for porcine haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and major acute phase protein (Pig-MAP) determinations. Intra and inter assay coefficients of variation (CVs) were lower than 20% in all cases with exception of inter assay CVs for CRP and Pig-MAP assays with samples of low acute phase proteins concentration, and for SAA assay at any acute phase proteins concentration. All methods showed good linearity and detection limits were low enough to detect APPs levels in healthy animals. Hp and SAA were very affected by haemolysis. Lipaemia influenced mainly on SAA determination. Over 15-fold increase was observed in CRP and SAA concentrations after artificially induced inflammation by a single subcutaneous dose of turpentine, whereas Hp and Pig-MAP increased less than 5-fold.