Antibodies to West Nile virus and related flaviviruses in wild boar, red foxes and other mesomammals from Spain

Red foxes (Vulpes vulpes), wild boar (Sus scrofa) and Iberian pigs (Sus scrofa domestica) that are raised extensively outdoors, as well as other wild mesomammals from south central Spain and wild boar from Do?ana National Park (DNP), were tested for antibodies against related flaviviruses by ELISA and for antibodies against WNV by VNT. Mean flavivirus seroprevalence according to ELISA was 20.4±7.8% (21 out of 103) in red foxes, 12.6±2.8% (69 out of 545) in wild boars, and 3.3±2.7% (6 out of 177) in Iberian pigs. A stone marten (Martes foina) also tested positive. Flavivirus seroprevalence in wild boar was significantly higher in DNP, and increased with age. Haemolysis of the serum samples limited interpretation of VNT to 28 samples, confirming WNV seroprevalence in one red fox, four Iberian pigs and nine wild boars. ELISA positive, microVNT negative samples suggest presence of non-neutralizing antibodies against WNV or antibodies to other antigenically related flaviviruses. Despite the importance of wetlands for flavivirus maintenance and amplification, WNV/flavivirus seroprevalence in wild boar and red foxes was not associated to wetland habitats. This is the first report of exposure of red foxes to WNV. With view to use of the tested species as sentinels for flavivirus activity, limited exposure of Iberian pigs that would be available for regular sampling, low numbers of foxes collected and concentration of wild boar harvest in the winter season are major drawbacks.     

Serological (Em2-ELISA) and parasitological examinations of fox populations for Echinococcus multilocularis infections

Serum or body fluid samples of 1,006 foxes were investigated in an ELISA for antibodies against a highly sensitive and specific antigen (Em2-antigen) of Echinococcus multilocularis. Parasitological examinations of the intestines and simultaneous serological examinations were carried out in 505 foxes: A group of 98 blue foxes (Alopex lagopus) from Norwegian fox farms did not contain intestinal stages of E. multilocularis and was clearly sero-negative in Em2-ELISA. On the other hand in red foxes (Vulpes vulpes) originating from European areas known to be endemic for E. multilocularis the following average prevalence rates were found: 244 foxes from Southern Germany, E. multilocularis prevalence 55% and sero-prevalence 60%; 139 foxes from Austria, E. multilocularis prevalence 4% and sero-prevalence 12%. Serological identification of individual foxes with or without intestinal E. multilocularis infection was not possible. Only serological (no parasitological) examination in 402 foxes originating from endemic areas in Switzerland resulted in a sero-prevalence rate of 37%. Sero-prevalence was only 6% and 4% in 54 and 26 other foxes, respectively, originating from Swiss and German areas where E. multilocularis has not yet been reported. Negative control Norwegian (farmed) silver foxes (n = 43) were all sero-negative. The specificity of the Em2-ELISA was confirmed by negative Em2-serologies with sera from dogs infected with intestinal and tissue dwelling helminth species (with the exception of two from 24 dogs infected with E. granulosus).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of muscle fluid and serum for detection of antibodies against hepatitis E virus in slaughter pigs

Muscle fluid was investigated as an alternative to serum for detecting anti-hepatitis E virus (HEV) antibodies in slaughter pigs. Samples of serum and diaphragmatic muscle juice from 67 pigs were analysed by anti-HEV IgG ELISA. Compared to the serum ELISA, the ELISA on diaphragmatic muscle fluid had a sensitivity of 93%, a specificity of 91.7%, a κ value of 0.84 and a determination coefficient (R(2)) of 0.77; both tests had global agreement of results. Muscle fluid can be used as an alternative to serum for serological detection of HEV antibodies in slaughter pigs.     

The ELISA for the examination of hare sera for anti-Brucella antibodies

Hare brucellosis is caused primarily by Brucella suis biovar 2. Hares along with wild boars are the natural reservoir of this microorganism. In view of restriction of applicability of traditional serological methods the work aimed to develop the ELISA to examine hare sera for the presence of anti-Brucella antibodies. Lipopolysaccharide (LPS) antigen obtained from the strain S19 of Brucella abortus and the conjugate of antibodies against rabbit immunoglobulin with horseradish peroxidase were used in the test. Hares' sera positive and negative in the CFT were used as controls of the ELISA. The sera collected from 9 hares suspected to be infected with Brucella organisms, positive in CFT (in this number 7 hares revealed clinical symptoms or anathomopathological lesions characteristic of brucellosis), 6 sera from hares showing no symptoms of the disease, negative in CFT and 520 sera from hares monitored for brucellosis were tested. All serum samples from hares suspected for Brucella infection were positive in ELISA and 2 of them were negative in RBPT. Additionally among the samples from hares monitored 12 sera were positive in ELISA and CFT, whereas 9 sera from 12 ones were also positive in the RBPT. The obtained results indicated that the ELISA developed in our laboratory proved to be equivalent in specificity to CFT. In addition, ELISA proved to be more sensitive than RBPT for the diagnosis of Brucella infection in hares.     

Evaluation of a novel enzyme-linked immunosorbent assay for serological diagnosis of porcine proliferative enteropathy

A specific enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the porcine pathogen Lawsonia intracellularis was developed and evaluated using sera from na?ve, naturally infected as well as experimentally infected pigs. On the basis of 37 serum samples collected from experimentally infected pigs and 62 serum samples from naturally infected pigs the sensitivity of the ELISA was calculated to 98.0%. The specificity of the test was 99.3%, calculated on the basis of 273 serum samples collected in six herds free of L. intracellularis after medicated eradication. The novel ELISA was a specific and sensitive method for detecting specific antibodies, and may be a good alternative to the existing serological tests for L. intracellularis. It may be usable for diagnosis of proliferative enteropathy and for determination of a herd's epidemiologic status.     

Evaluation of tongue inspection and serology for diagnosis of Taenia solium cysticercosis in swine: usefulness of ELISA using purified glycoproteins and recombinant antigen

Evaluation of serology using glycoproteins (GPs) purified by preparative isoelectric focusing (pH 8.8) and recombinant chimeric antigen (RecTs) of Taenia solium was carried out using (1) blood samples on filter papers from pigs infected with different doses of eggs of T. solium in Mexico, (2) serum samples from pigs found infected naturally in Vietnam and Ecuador and (3) serum samples from pigs suspected to be infected with T. solium by tongue inspection in Tanzania. Antibody responses (IgG) were detectable in experimentally infected pigs confirmed harbouring 16 or more cysts at necropsy from 30 days after egg inoculation. One of three pigs naturally infected and harbouring 2.5 cysts/kg muscle and most of pigs harbouring=5.0 cysts/kg were also seropositive by ELISA. Although pigs may be infected with other taeniid species such as Taenia hydatigena, pigs harbouring this parasite were negative in ELISA. Approximately, 76 and 78% of sera from pigs having nodule(s) in the tongue (positive tongue inspection) were serologically positive by both ELISA and immunoblot, respectively. Furthermore, approximately 34 and 18% of sera from pigs having no nodules in the tongue (negative tongue inspection) were also seropositive by ELISA and immunoblot, respectively. ELISA using the two antigens was more sensitive than immunoblot and reliable for differentiation of pigs infected with cysticerci of T. solium from those either uninfected or infected with other taeniid species. Pigs without nodule by tongue inspection should be checked serologically in endemic areas.     

A serological survey of porcine reproductive and respiratory syndrome virus in wild boar in Gifu Prefecture,Japan

Porcine reproductive and respiratory syndrome (PRRS) is an infectious swine disease caused by the PRRS virus (PRRSV) that results in economic loss to the pig-rearing industry. To study PRRSV infection in wild boars and pigs, we conducted a serological survey in Gifu Prefecture, Japan, from 2020 to 2021. Three out of 453 (0.7%) wild boar sera were positive for PRRSV antibodies in a commercial ELISA. However, given that PRRSV RNA was not detected in these three wild boars and the specificity and sensitivity of the test kit, these are considered as false positives. Although seropositive pigs were found in multiple pig farms in the study area, the role of wild boars as a source of PRRS to pig farms appeared to be minimal.     

Prevalence of porcine circoviruses in Transylvanian wild boars, detected by real-time PCR--short communication

Porcine circoviruses (PCV) are widespread in domestic pigs worldwide and there is growing information about the presence of PCV in other suid species. Based on serological studies with sera of wild boars, it was established that PCV1 was present in these animals and antibodies specific to PCV2 were also detected in wild boars living in captivity or in sylvatic areas, both with or without clinical signs of PMWS. Studies including PCV2 genome or antigen detection confirmed the previous findings. This is the first report about the presence of PCV in Transylvanian wild boar populations. Four hundred and sixty-nine samples were collected and grouped according to geographic origin, tested for the presence of PCV DNA using a real-time quantitative polymerase chain reaction assay, and 13.52% of the animals proved to be positive for one or in three cases both of the PCV genotypes. PCV2 was detected in all of the PCV-positive samples.     

Prevalence of Trichinella spp. in red foxes and wild boars in the northwestern part of Poland

The aim of the study was to establish in which degree wild boars and red foxes are reservoir of Trichinella spp. in North-West Poland. Research was carried out between 1997 and 2004 on 505 foxes and 56,462 wild boars in muscle samples. The muscle samples were examined using the digestion method. The average prevalence rate of Trichinella spp. infection of foxes was 4.4 %. Large differences of the infection rate in wild boars were observed. In the years 1999-2001 Trichinella spp. larvae were observed in 58 animals (0.2 %) and between 2002 and 2004 the Trichinella spp. prevalence in 227 wild boars was 0.9 %, demonstrating that the animals were 5.1 times more often infected than in 1999-2001. The growth of red fox population after the oral vaccination against rabies was probably the cause of this phenomenon.     

Potential application of serological tests on fluids from carcasses: detection of antibodies against Toxoplasma gondii and Sarcoptes scabiei in red foxes (Vulpes vulpes)

Background

Serological surveys for disease investigation of wild animal populations require obtaining blood samples for analysis, which has logistic, ethic and economic difficulties. Applying serological test to fluids collected from dead animals is an alternative. The aim of this study was to assess if antibodies could be detected in two types of fluids collected from 56 carcasses of red foxes (Vulpes vulpes): pleural fluid and lung extract.

Findings

In 22 (39%) foxes antibodies against Sarcoptes scabiei were detected in both fluid types by ELISA and Western blot. In 46 (82%) foxes, antibodies against Toxoplasma gondii were detected in pleural fluid and in 41 (73%) in lung extract applying a Toxo-screen test (DAT). Antibodies were still detectable in the same fluids kept at room temperature for 28 days, although in fewer foxes (16 and 14 foxes tested for T. gondii in lung extract and pleural fluid respectively; and 1 and 4 tested for S. scabiei in lung extract and pleural fluid respectively.

Conclusions

These results indicate the potential utility of using fluids from carcasses for antibody screening of wild animals at the population level.     

Comparison of two antigens for use in an enzyme-linked immunosorbent assay to detect African swine fever antibody

Two African swine fever virus (ASFV) antigens were tested for use in an ELISA to detect antibody to ASFV. Antigens used were the cytoplasmic soluble fraction (CS-P) of infected cells grown in the presence of porcine serum and the semipurified viral structural protein VP73 (SVP73). Both antigens were tested by ELISA against 72 sera obtained during several ASF field episodes and from ASFV-inapparent carriers. Of the 72 sera, only 2.8% has positive results by ELISA against CS-P antigen; 60% of positive-reacting sera (to both antigens) had higher ELISA values when the CS-P antigen was used. Samples (with positive results) that reacted only to CS-P antigen had results confirmed by immunoblot analysis. Such sera reacted against ASFV-infection proteins IP25, IP25.5, and IP30, but not against IP73. In time-course experiments to detect appearance of ASFV-antibodies in infected miniature pigs, antibodies were detected by immunoblot analysis on postinoculation day (PID) 8. At that time, only the polypeptides IP25, IP25.5 IP30, and IP31 were recognized; IP73 and IP12 were first detected 3 and 4 days later, respectively. In the same experiments, ASFV antibodies were detected by ELISA, using CS-P or SVP73 antigens, on PID 7 and 9, respectively. These results could explain the percentage of sera not having positive results by ELISA using SVP73 antigen, if the sera were obtained from ASFV-infected pigs during the first days of infection before induction of antibody response against the IP73 protein. This feature makes the use of CS-P antigen advantageous in early serologic detection of ASFV-infected pigs.     

Survey for Trichinella spp. in red foxes (Vulpes vulpes) in Belgium.

Concurrently with a survey for Echinococcus multilocularis in the red fox (Vulpes vulpes) in Flanders, northern Belgium, serological and parasitological analyses for Trichinella spp. were carried out from 1996 to 1999. Muscle samples from foxes in Wallonia, southern Belgium, were obtained during a survey for rabies and alveolar echinococcosis from 1998 to 2000.In muscle samples from tongue, diaphragm, hindlegs and tail of 179 Flemish foxes no larvae were found by trichinoscopy. Serum and muscle juice of, respectively 176 and 26 animals were examined using an ELISA for the detection of antibodies against excretory-secretory (ES) antigen. There were eight (4.5%) positive sera, but no positive muscle juice samples.All muscle samples from 639 foxes in Wallonia proved to be negative for larvae in artificial digestion. Serum and muscle juice of 130 and 478 foxes, respectively were examined in ES-ELISA. There were 61 (46.9%) positive sera and 90 (18.8%) positive muscle juice samples. A comparison between 88 serum and muscle juice samples of the same foxes showed that only half of the serum-positive animals were detected using muscle juice. However, for establishing the true meaning of these results, a more profound epidemiological study on the vulpine population in Belgium is necessary.     

Seroprevalence of Hepatitis E Virus in Domestic Pigs and Wild Boars in Switzerland

Hepatitis E is considered an emerging human viral disease in industrialized countries. Studies from Switzerland report a human seroprevalence of hepatitis E virus (HEV) of 2.6–21%, a range lower than in adjacent European countries. The aim of this study was to determine whether HEV seroprevalence in domestic pigs and wild boars is also lower in Switzerland and whether it is increasing and thus indicating that this zoonotic viral infection is emerging. Serum samples collected from 2,001 pigs in 2006 and 2011 and from 303 wild boars from 2008 to 2012 were analysed by ELISA for the presence of HEV‐specific antibodies. Overall HEV seroprevalence was 58.1% in domestic pigs and 12.5% in wild boars. Prevalence in domestic pigs was significantly higher in 2006 than in 2011. In conclusion, HEV seroprevalence in domestic pigs and wild boars in Switzerland is comparable with the seroprevalence in other countries and not increasing. Therefore, prevalence of HEV in humans must be related to other factors than prevalence in pigs or wild boars.     

Detection of specific antibodies in dogs infected with Angiostrongylus vasorum

Canine angiostrongylosis, caused by the nematode Angiostrongylus vasorum, is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We determined the diagnostic value of the specific detection of antibodies against A. vasorum adult somatic antigen, adult excretory/secretory (E/S) antigen and first stage larvae (L1) somatic antigen in ELISAs. Also, A. vasorum adult somatic antigen purified by monoclonal antibodies (mAb) was evaluated in a sandwich-ELISA. Among the crude antigens, the best sensitivities when testing 21 naturally infected dogs were obtained using adult E/S and somatic antigen (85.7% and 76.2%, respectively), which were comparable with the results of the sandwich-ELISA based on mAb-purified antigens (81%). The ELISA performed with L1 antigen had the lowest sensitivity (42.9%). In experimentally inoculated dogs, the sensitivities ranged from 97.7% to 100% with all test settings. The specificity was 98.8% (92.5-99.9%, 95% CI) with all ELISAs using sera of 82 randomly selected dogs. Cross-reactions using adult somatic, adult E/S and L1 somatic antigen were observed in sera of dogs infected with Crenosoma vulpis, Dirofilaria immitis, Dirofilaria repens, and Eucoleus aerophilus. In contrast, using the mAb-purified antigens, the cross-reactions were minimal. Depending on the antigens used, specific antibodies were detected starting between 13 and 21 days post experimental inoculation (dpi), and at latest between 35 and 48 dpi, thus before or around the onset of patency. The serological follow-up of four A. vasorum-infected dogs after anthelmintic treatment at 88 dpi showed a decrease of antibody levels after drug administration, and the animals became seronegative 2-9 weeks later. Two untreated dogs remained seropositive. In four dogs treated 4 dpi, virtually no antibody-reaction was detectable, with the exception of the ELISA performed with L1 antigen. The early detection of specific antibodies against A. vasorum by ELISA represents a valid alternative for a reliable diagnosis and for follow-up investigations after anthelmintic treatment.     

Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera

Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.     

Classical swine fever virus: clinical, virological, serological and hematological findings after infection of domestic pigs and wild boars with the field isolate "Spante" originating from wild boar

A classical swine fever virus (CSFV) field isolate originating from wild boar was investigated on its virulence in domestic pigs and wild boar. Three weaner pigs and two wild boars (yearlings) were intranasally inoculated with the isolate "Spante" and tested for clinical, virological, hematological and serological findings until day 31 after infection (p. i.). One day p. i. the piglets were put in contact to three sentinel pigs. During a period of 31 d neither the domestic pigs nor the wild boars showed clinical signs specific for CSF. Two infected weaner pigs became transiently viraemic, transmitted CSFV in nasal secretions, showed a slight leukopenia and reacted serologically positive. The contact infection resulted in a viraemia in two sentinel piglets on day 30. Only one contact animal developed antibodies. None of the wild boars became viraemic, excreted CSFV in nasal secretions or developed antibodies. The CSFV isolate "Spante" represents a low virulent virus. Referring to a significant higher percentage of virologically positive tissue samples after nested PCR compared with the virus isolation, persistence of CSFV is discussed.     

Indirect micro-enzyme-linked immunosorbent assay for the detection of antibodies to Mycoplasma synoviae and M. gallisepticum

The sensitivity and specificity of the indirect micro-enzyme-linked immunosorbent assay (ELISA) was compared with that of the rapid serum-plate test (RSPT) and the hemagglutination-inhibition test (HIT) in detecting antibodies to Mycoplasma gallisepticum (MG) and M. synoviae (MS). Membrane antigens of MG strain S6 and MS strain NEL 61800 were used. ELISA was performed with single MS and single MG antigens and a combined MS/MG antigen. The MS-ELISA was as sensitive as the MS-RSPT and more sensitive than and as specific as the MS-HIT in detecting antibodies to MS. The MG-ELISA was less sensitive than the MG-RSPT and slightly less sensitive than the MG-HIT in detecting antibodies to MG in chickens experimentally infected with MG R strain but more sensitive in detecting antibodies in chickens infected with MG F strain. MG-ELISA resulted in fewer cross-reactions than the MG-RSPT but more than the MG-HIT. The combined MG/MS-ELISA was as sensitive as the ELISA with its individual antigen components. No nonspecific reactions were observed with sera from MG/MS-free flocks. The combined MG/MS-ELISA was found to be a practical screening test for antibodies to both MS and MG. Further improvement of the sensitivity and the specificity of the MG antigen is desirable.     

Comparative assessment of a double antibody enzyme immunoassay test kit and a triple antibody enzyme immunoassay for the diagnosis of Trichinella spiralis spiralis and Trichinella spiralis nativa infections in swine.

Enzyme immunoassays using the triple antibody enzyme linked immunosorbent assay (ELISA) with both Trichinella spiralis spiralis and T. spiralis nativa excretory-secretory (ES) antigens and a commercial Trichinella spiralis enzyme immunoassay test kit were carried out on sera from pigs that were infected with light, moderate and high doses of infective T. spiralis spiralis and T. spiralis nativa respectively. Seroconversion occurred in all pigs given infective Trichinella larvae although no trichinae were recovered from pigs given T. spiralis nativa larvae and examined between days 92 and 99 postinfection by pepsin digestion. Anti-Trichinella antibodies were detected in pigs infected with T. spiralis spiralis and T. spiralis nativa by ELISA using either the homologous or heterologous ES antigen. The commercial Trichinella spiralis enzyme immunoassay test kit also detected anti-Trichinella antibodies in both the T. spiralis spiralis and T. spiralis nativa infected pigs. The commercial test kit did not appear to be as sensitive as the triple antibody ELISA since it usually took two to three days longer for seroconversion to be detected by the former procedure. Finally seroconversion occurred more rapidly in swine infected with T. spiralis spiralis than with pigs receiving comparable doses of T. spiralis nativa.     

Effect of parasite burden on the detection of Fasciola hepatica antigens in sera and feces of experimentally infected sheep

The effect of Fasciola hepatica parasite burden on the detection of excretory/secretory (E/S) antigens in sera and feces of experimentally infected sheep was evaluated using a double antibody-based capture enzyme-linked immunosorbent assay (ELISA). Four groups of five sheep each were used. The first three groups were infected with 50, 100 and 200 metacercariae of F. hepatica, and the fourth group remained as non-infected control. On the day of infection and weekly thereafter, serum and fecal samples were taken. ELISA detected F. hepatica E/S antigen levels in serum from the first week post-infection (wpi) and in fecal supernatant from the fourth wpi, which were significantly (p<0.05) higher than controls. F. hepatica eggs were not detected until after the eighth wpi. The correlation between absorbance of E/S antigens in serum with the fluke burden was 0.77 (p<0.0001) and in feces 0.76 (p<0.0001) at 12th wpi. The sensitivity of the assay to detect E/S antigens in serum was 86.6% and in feces 93.3%. It is concluded that the ELISA technique used in this study offers a diagnostic alternative for detecting early infections of F. hepatica in sheep.     

A serosurvey of Hepatozoon canis and Ehrlichia canis antibodies in wild red foxes (Vulpes vulpes) from Israel

A seroepidemiological survey was conducted to investigate the prevalence of antibodies reactive with Ehrlichia canis and Hepatozoon canis antigens in free-ranging red foxes (Vulpes vulpes) in Israel. Of 84 fox sera assayed, 36% were seropositive for E. canis by the indirect fluorescent antibody (IFA) test and 24% were positive for H. canis using an enzyme-linked immunosrbent assay (ELISA). Canine ehrlichiosis and hepatozoonosis appear to be endemic in the wild red fox populations in Israel, and foxes may serve as a reservoir for infection of domestic dogs and other wild canine species.