Experimental Infection of Domestic Pigs with African Swine Fever Virus Lithuania 2014 Genotype II Field Isolate.

An experimental infection was conducted to evaluate horizontal transmission, clinical, virological and humoral response induced in domestic pigs infected with African swine fever (ASF) genotype II virus circulating in 2014 into the European Union (EU). Ten naive pigs were placed in contact with eight pigs experimentally inoculated with the Lithuanian LT14/1490 ASF virus (ASFV) responsible for the first ASF case detected in wild boar in Lithuania in January 2014. Clinical examination and rectal temperature were recorded each day. Blood sampling from every animal was carried out twice weekly. Blood samples were examined for presence of ASF virus-specific antibodies and for determining the ASFV viral load. From the obtained results, it was concluded that the Lithuanian ASFV induced an acute disease which resulted in 94, 5% mortality. The disease was easily detected by real-time PCR prior to the onset of clinical signs and 33% of the animals seroconverted. All findings were in accordance with observations previously made in domestic pigs and wild boar when infected with ASF genotype II viruses characterized by a high virulence. One in-contact pig remained asymptomatic and survived the infection. The role of such animals in virus transmission would need further investigation.

Experimental Transmission of African Swine Fever (ASF) Low Virulent Isolate NH/P68 by Surviving Pigs.

African swine fever (ASF) has persisted in Eastern Europe since 2007, and two endemic zones have been identified in the central and southern parts of the Russian Federation. Moderate- to low-virulent ASF virus isolates are known to circulate in endemic ASF-affected regions. To improve our knowledge of virus transmission in animals recovered from ASF virus infection, an experimental in vivo study was carried out. Four domestic pigs were inoculated with the NH/P68 ASF virus, previously characterized to develop a chronic form of ASF. Two additional in-contact pigs were introduced at 72 days post-inoculation (dpi) in the same box for virus exposure. The inoculated pigs developed a mild form of the disease, and the virus was isolated from tissues in the inoculated pigs up to 99 dpi (pigs were euthanized at 36, 65, 99 and 134 dpi). In-contact pigs showed mild or no clinical signs, but did become seropositive, and a transient viraemia was detected at 28 days post-exposure (dpe), thereby confirming late virus transmission from the inoculated pigs. Virus transmission to in-contact pigs occurred at four weeks post-exposure, over three months after the primary infection. These results highlight the potential role of survivor pigs in disease maintenance and dissemination in areas where moderate- to low-virulent viruses may be circulating undetected. This study will help design better and more effective control programmes to fight against this disease.

Assessment of African Swine Fever Diagnostic Techniques as a Response to the Epidemic Outbreaks in Eastern European Union Countries: How To Improve Surveillance and Control Programs.

This study represents a complete comparative analysis of the most widely used African swine fever (ASF) diagnostic techniques in the European Union (EU) using field and experimental samples from animals infected with genotype II ASF virus (ASFV) isolates circulating in Europe. To detect ASFV, three different PCRs were evaluated in parallel using 785 field and experimental samples. The results showed almost perfect agreement between the Universal ProbeLibrary (UPL-PCR) and the real-time (κ = 0.94 [95% confidence interval {CI}, 0.91 to 0.97]) and conventional (κ = 0.88 [95% CI, 0.83 to 0.92]) World Organisation for Animal Health (OIE)-prescribed PCRs. The UPL-PCR had greater diagnostic sensitivity for detecting survivors and allows earlier detection of the disease. Compared to the commercial antigen enzyme-linked immunosorbent assay (ELISA), good-to-moderate agreement (κ = 0.67 [95% CI, 0.58 to 0.76]) was obtained, with a sensitivity of 77.2% in the commercial test. For ASF antibody detection, five serological methods were tested, including three commercial ELISAs, the OIE-ELISA, and the confirmatory immunoperoxidase test (IPT). Greater sensitivity was obtained with the IPT than with the ELISAs, since the IPT was able to detect ASF antibodies at an earlier point in the serological response, when few antibodies are present. The analysis of the exudate tissues from dead wild boars showed that IPT might be a useful serological tool for determining whether or not animals had been exposed to virus infection, regardless of whether antibodies were present. In conclusion, the UPL-PCR in combination with the IPT was the most trustworthy method for detecting ASF during the epidemic outbreaks affecting EU countries in 2014. The use of the most appropriate diagnostic tools is critical when implementing effective control programs.

Comparative evaluation of novel African swine fever virus (ASF) antibody detection techniques derived from specific ASF viral genotypes with the OIE internationally prescribed serological tests.

The presence of antibodies against African swine fever (ASF), a complex fatal notifiable OIE disease of swine, is always indicative of previous infection, since there is no vaccine that is currently used in the field. The early appearance and subsequent long-term persistence of antibodies combined with cost-effectiveness make antibody detection techniques essential in control programmes. Recent reports appear to indicate that the serological tests recommended by the OIE for ASF monitoring are much less effective in East and Southern Africa where viral genetic and antigenic diversity is the greatest. We report herein an extensive analysis including more than 1000 field and experimental infection sera, in which the OIE recommended tests are compared with antigen-specific ELISAs and immuno-peroxidase staining of cells (IPT). The antibody detection results generated using new antigen-specific tests, developed in this study, which are based on production of antigen fractions generated by infection and virus purification from COS-1 cells, showed strong concordance with the OIE tests. We therefore conclude that the lack of success is not attributable to antigenic polymorphism and may be related to the specific characteristics of the local breeds African pigs.

Molecular diagnosis of African Swine Fever by a new real-time PCR using universal probe library.

A highly sensitive and specific real-time PCR method was developed for the reliable and rapid detection of African swine fever virus (ASFV). The method uses a commercial Universal Probe Library (UPL) probe combined with a specifically designed primer set to amplify an ASFV DNA fragment within the VP72 coding genome region. The detection range of the optimized UPL PCR technique was confirmed by analysis of a large panel (n = 46) of ASFV isolates, belonging to 19 of the 22 viral p72 genotypes described. No amplification signal was observed when closely clinically related viruses, such as classical swine fever, or other porcine pathogens were tested by this assay. The detection limit of the UPL PCR method was established below 18 DNA copies. Validation experiments using an extensive collection of field porcine and tick samples (n = 260), coming from Eastern and Western African regions affected by ASF, demonstrated that the UPL PCR technique was able to detect over 10% more positive samples than the real-time TaqMan PCR test recommended in the OIE manual, confirming its superior diagnostic sensitivity. Clinical material collected during experimental infections with different ASFV p72 genotypes was useful for assuring both the capacity of the UPL PCR for an early viral DNA detection and the competence of the technique to be applied in any ASF diagnostic target sample. The reliability and robustness of the UPL PCR was finally verified with a panel of ASFV-infected clinical samples which was repeatedly tested at different times. Additionally, an internal control PCR assay was also developed and standardized using UPL probes within the endogenous ß-actin gene. Finally, the complete study offers a new validated real-time PCR technique, by means of a standardized commercial probe, providing a simple, rapid and affordable test, which is ready for application in the routine diagnosis of ASF.

Comparison of African swine fever virus prevalence and risk in two contrasting pig-farming systems in South-west and Central Kenya.

We describe a horizontal survey of African swine fever virus (ASFV) prevalence and risk factors associated with virus infection in domestic pigs in two contrasting production systems in Kenya. A free range/tethering, low input production system in Ndhiwa District of South-western Kenya is compared with a medium input stall fed production system in Kiambu District of Central Kenya. Analysis of variance (ANOVA) of data derived from cluster analysis showed that number of animals, number of breeding sows and number of weaner pigs were a significant factor in classifying farms in Nhiwa and Kiambu. Analysis of blood and serum samples using a PCR assay demonstrated an average animal level positivity to ASFV of 28% in two independent samplings in South-western Kenya and 0% PCR positivity in Central Kenya. No animals were sero-positive in either study site using the OIE indirect-ELISA and none of the animals sampled exhibited clinical symptoms of ASF. The farms that contained ASFV positive pigs in Ndhiwa District were located in divisions bordering the Ruma National Park from which bushpig (Potamochoerus larvatus) incursions into farms had been reported. ASFV prevalence (P<0.05) was significantly higher at distances between 6 and 16km from the National Park than at distances closer or further away. One of the 8 bushpigs sampled from the park, from which tissues were obtained was PCR positive for ASFV. The data therefore indicated a potential role for the bushpig in virus transmission in South-western Kenya, but there was no evidence of a direct sylvatic virus transmission cycle in Central Kenya. ASF control strategies implemented in these areas will need to take these epidemiological findings into consideration.

The effect of anti-Anisakis simplex antibody levels on C3 and C4 complement components in human sera.

Previously, an in vitro effect was observed on the complement system not only of the excretory-secretory products but also of somatic antigens from L3 Anisakis simplex larvae. In the present work the effect of anti-A. simplex specific antibodies on C3 and C4 levels in human sera was investigated. Up to 309 samples of sera were tested to determine levels of C3 and C4 and anti-A. simplex antibodies, including immunoglobulins IgG, IgM, IgA and IgE. Significant differences were observed between levels of C3 and C4 and all immunoglobulins except for IgE. In the case of immunoglobulins, the probability that an anti-A. simplex positive subject has a C3 deficiency was 3.8 times higher than a subject without specific antibodies. In conclusion, an association between elevated levels of anti-A. simplex antibodies and C3 and C4 deficiency was demonstrated.

Blood parameters are little affected by time of sampling after the application of ketamine in black howler monkeys (Alouatta pigra).

BACKGROUND: Ketamine hydrochloride is an anesthetic commonly utilized to obtain biological samples in various non-human primates. Its application alters individual hematologic and biochemical values. The aim of this study was to analyze its effect on blood parameters of Alouatta pigra. METHOD: We collected blood samples at 10 and 40 minutes after the application of ketamine in 12 adult female A. pigra living in free-ranging conditions. RESULTS: The analysis showed that 40 minutes after application of ketamine, the number of platelets, lymphocytes and concentration of phosphorus decreased; however, creatinine, cholesterol, triglycerides, and potassium values increased. CONCLUSIONS: Our results suggest that ketamine appears to have little effect on the hematology and blood biochemistry of Alouatta pigra females with respect to those reported for other non-human primates. It is also important to consider the elapsed time after their application when taking blood samples for proper interpretation of the hemogram of Alouatta pigra females.

Surveillance for African swine fever in Nigeria, 2006-2009.

African swine fever (ASF) has had significant economic and social impact in Nigeria since 1997. However, there has been no effective national response to bring it under control. In this report, we confirm that ASF is still prevalent and widespread in Nigeria. Results from both serosurveillance and virological analyses indicated that ASF is present in most of the agro-ecological zones of the country. Nine per cent (9%) of serum samples and 48% of tissue samples were positive for ASF virus antibody and genome, respectively. Areas with high pig-related activities (marketing, consumption and farming) have higher prevalences compared with areas with less pig activities. Farm-gate buyers, marketing systems and transport of untested pigs within the country assist with the circulation of the virus. Only by putting in place a comprehensive routine surveillance and testing system, reorganizing the market and transportation systems for pigs, implementing on-farm bio-security protocols and considering the option of compensation will it be possible to achieve a significant reduction in ASF prevalence in Nigeria.

Differential effect of appendectomy and tonsillectomy on anti-Kudoa sp. antibodies in patients with MALTectomy.

We found an association between tonsillectomized patients and subsequent appendicitis. We also observed that MALTectomy significantly decreased secretory IgA levels in serum of patients, being this decrease more pronounced when both operations (tonsillectomy and appendectomy) had been performed. The elevated humoral responses detected previously by us in BALB/c mice immunized with Kudoa sp. pseudocyst extracts and the high IgG1 and IgE levels induced by the oral administration of Kudoa sp. pseudocysts to BALB/c mice showed the possible immunopathological effects in man from the ingestion of Kudoa sp. infected fish. We use the ELISA method to investigate the possible relationship between MALTectomy (tonsillectomy and appendectomy) and specific antibody levels to Kudoa sp. Both anti-Kudoa sp. specific antibody levels and the number of patients that recognized Kudoa sp. antigens were greater in tonsillectomy patients when compared to the control and the other studied groups (appendectomized and appendectomized+tonsillectomies patients). Tonsillectomy was associated to a switch in the class of immunoglobulins involved in these responses and these responses may be abrogated by appendectomy. Tonsils and appendix may respond in different ways to Kudoa sp. antigens and these different reactions may be involved in some immunopathological reactions.

Seroprevalence of anti-Gymnorhynchus gigas (Trypanorhyncha, Gymnorhynchidae) antibodies in a Spanish population.

The somatic products released from ingested larvae of Gymnorhynchus gigas parasitizing fish induce a Th2 response capable of causing allergic disorders. The objective of the present work was to evaluate the prevalence of anti-Gymnorhynchus gigas antibodies in a Spanish population and established a possible relationship with fish consumption habits. We studied 305 residents in Madrid, with neither clinical symptoms suggestive of gastrointestinal or allergic disorders, nor pathologies related to ingestion of fish that could cause disease. Specific antibody levels were measured by ELISA: 11.8%, 20%, 15.7%, 21%, and 7.5% of the total studied sera were IgA, Ig's, IgG, IgM, and IgE positive, respectively. Seropositivity was not more prevalent among fresh fish consumers and did not increase with frequency of fish consumption. IgE values were lower in the group that never ingested smoked fish. Anti-G. gigas antibody levels were higher in the group that reported frequent consumption of marinated fish. The use of cooking methods with the least heating efficacy (frying, or frying in batter, and microwaving) did not affect seropositivity percentages among consumers. Infection with live plerocercoids is not necessary for seropositivity, and the antibody production, in this case, is due to the absorption of antigens from the parasite following the digestion process. The human health risks of allergic reactions due to parasite antigens remain active after freezing the fish.

Anisakis simplex and Kudoa sp.: evaluation of specific antibodies in appendectomized patients.

High prevalence and intensity of infection with anisakid larvae has been reported in commercially important fish in Spain. Likewise, Kudoa-infected fish have lately been detected in both fresh and frozen fish. In the present study the possible relation between appendectomy and specific antibodies to these fish parasites was investigated. One hundred and sixty patients were enrolled in this study. They were divided into two groups of eighty patients each and matched for sex and age: Group 1 (appendectomized) and Group 2 (control group). Total immunoglobulins (Ig's), IgG, IgM, IgA and IgE against Anisakis simplex or Kudoa sp. antigens were analysed by ELISA. The mean values of the specific antibodies were lower in the appendectomy group, although significant differences were not observed in the case of IgG, IgA and IgE anti-A. simplex and IgE anti-Kudoa sp. In summary, appendectomy significantly decreased serum specific immunoglobulin levels against these food borne parasite antigens. This decrease was detectable from three months to three years post-appendectomy. It is necessary to study the influence of the surgical removal of other important parts of the GALT on these anti-parasite humoral immune responses.