SHOULD WE USE ANTI-GEN OR ANTI-BODY TESTS?
Recently, I came across a publication from Pr Sun & al confirming the circulation of a less virulent strain of African Swine Fever in Asia. What could appear as good news is actually describing a worrying situation as contaminated animals are surviving and shedding virus for months instead of only weeks for the acute form as such acute form ultimately kill most animals. That means that if this less virulent virus starts circulating, it is getting more and more difficult to get rid of that disease.
African swine fever (ASF) is a highly contagious, hemorrhagic swine disease caused by the African swine fever virus (ASFV). The disease continues to pose a major threat to the pig industry, food security, and rural development worldwide and namely in Asia, and is listed as a notifiable disease by the World Organization for Animal Health (OIE).
The disease signs and mortality rates of ASF vary among viral strains and animal species. The acute disease presents with high fever, depression, hemorrhages, cyanosis, and death within 15 days with near 100% mortality.
ASF was first described in Kenya in 1921 and spread throughout sub-Saharan Africa. Genotype I virus was first found in Portugal outside Africa in 1957 and caused the acute onset of infection; it then appeared in Spain, France, Madeira, Italy, Cuba, Malta, Sardinia, Brazil, the Dominican Republic, and Haiti in the 1960s and 1970s. Genotype I ASFVs have since been eradicated in all these countries except Italy-Sardinia, where it has been endemic since 1978.
In 2007, genotype II ASFV was first introduced into Georgia outside Africa and then spread to other Caucasian and European countries. In 201..8, Georgia-07-like genotype II ASFV emerged in China and spread to 15 other Asian countries. This virus causes very acute disease with near 100% mortality and has been prevalent in China and South-East Asia for almost 3 years.
In June 2021, a fattening pig weighing about 80 Kg showed paralytic symptoms on a farm in Shandong province and was euthanized for autopsy. The lung sample was collected and delivered to the Chinese National African Swine Fever Para-reference Laboratory (CNASFPL) for ASFV detection. On another farm in Henan province, the fattening pigs developed chronic infection signs including weight loss, intermittent fever, skin ulcers, and arthritis; sporadic deaths were also observed. Samples from four dead pigs, including lymph nodes and spleens, were analyzed. All samples were confirmed to be ASFV positive. Further sequence analysis of the p72 genes indicated that the ASFVs in these samples belonged to genotype I.
Since 2018, Georgia-07-like genotype II ASFVs with high virulence has been prevalent in China. Now, genotype I epidemic strains with lower virulence than attenuated genotype II viruses have re-emerged in the field. Clinical signs begin 14 to 21 days post-infection with a slight fever, followed by mild respiratory distress and moderate-to-severe joint swelling. This is often combined with reddened areas of skin that become raised and necrotic. Additional necropsy findings include pneumonia with caseous necrosis (sometimes with focal mineralization) in lungs, fibrinous pericarditis, and edematous lymph nodes, which can be partially hemorrhagic (mainly mediastinal lymph nodes). Chronic forms often result in lethality rates that are typically less than 30 percent.
Infected pigs continuously shed viruses and develop low-level viremia, which makes an early diagnosis by antigen tests more difficult than for attenuated genotype II viruses in the field.
The re-emergence of the genotype I ASFVs will cause more problems and pose bigger challenges for ASF eradication in Asia. The newly emerging genotype I ASFVs may cause severe and continuous economic losses to the pig industry, once they spread in swine herds or infect breeding sows and boars.
Clinical diagnosis can be difficult during the early stages of the disease, or when small numbers of animals are affected. Diagnosing ASF is often speculative, for symptoms may be confused with those of other diseases like Classical Swine Fever, PRRS, Erysipelas, and even septicemia like Salmonellosis. No diagnosis is conclusive until confirmed by antigen or antibody diagnostic tests. A positive test for the presence of the virus (i.e. antigenic tests like PCR test) indicates that the tested animal is undergoing infection at the time of sampling but a negative result from an antigenic test does not enable to conclude whether the animal had been contaminated in the past and maybe already recovered. The only way to conclude on past contamination of pigs would be to confirm the presence of antibody as it remains seropositive for life.
As long as ASF is an acute disease with high mortality, antigen tests are the only relevant tests as animals die anyway few days after infection and before the production of antibodies. But as the chronic form is developing, the presence of virus in the blood may not be sufficient anymore to conclude and the confirmation of antibody level may become more relevant information to understand past months infection status of animals.
Since late 2015, epidemiological serological data in Eastern Europe has shown a significant increase in the incidence of seropositive animals, particularly evident in wild boar populations in the affected EU countries. These results suggest that some animals are surviving for over a month, may be able to recover from ASF infection, and in certain cases, even remain sub-clinically infected, as previously described in the Iberian Peninsula, the Americas, and in Africa. Antibody detection techniques are therefore essential to obtain complete information in support of control and eradication programs.
Antigen tests
As far as antigen tests are concerned, Polymerase Chain Reaction (PCR) is used to detect the ASFV genome in porcine samples (blood, organs, etc.) and ticks (vectors collected in the farm). All validated PCR tests allow viral DNA detection even before the appearance of clinical signs. PCR enables the diagnosis of ASF to be made within hours of sample arrival to the laboratory. PCR provides a sensitive, specific, and rapid alternative to virus isolation for the detection of ASFV. PCR provides higher sensitivity and specificity than alternative methods for antigen detection, such as the antigen enzyme-linked immunosorbent assay (ELISA) and the direct fluorescent antibody test (FAT) but it takes time, requires equipment, and is quite expensive.
Viral antigens can also be detected using ELISA, which is cheaper to set up than PCR methods and allows large-scale testing of samples in a short time without special laboratory equipment. However, in subacute and chronic diseases, the antigen ELISA has a significantly decreased sensitivity. In addition, field samples are often in poor condition and therefore also decrease the sensitivity of the test. It is thus recommended to use the antigen ELISA (or any other ELISA) only as a “herd” test and in conjunction with other virological and serological tests.
Antibody tests
They are required to confirm breeders’ status before entering the farm or to release fatteners before slaughtering. ASFV antibodies appear about 7-9 days post-infection and can be detected for the rest of the animal’s life.
For the detection of ASF antibodies, the recommended tests include the ELISA test for anti-body screening. The ELISA test is a very useful technique, widely used for large-scale serological studies of many animal diseases. Some of the most notable characteristics of this method are high sensitivity (low false negative) and specificity (low false positive) indexes, high speed, low cost, and easy interpretation of results. Large populations can be rapidly screened thanks to the automatic equipment available.
But antibody ELISA sensitivity and specificity can be affected by the handling of the blood samples. Indeed, in case of incorrectly handled or badly preserved (due to inadequate storage or transportation), the false-positive results can increase up to 20%. Moreover, the purity of the protein selected by test manufacturers can affect the test sensitivity. If the protein selected is mixed with other proteins, that could get positive reactions with other antibodies and increase the portion of false positive. Such effects on the sensitivity and specificity ratio of the test used could have a major impact on the industry. Higher false positive would lead to unnecessary elimination whether false negative could let contaminated carcasses sold on the meat market.
Therefore, all positive and doubtful samples by ELISA must be confirmed by alternative serological confirmatory tests.
Recently a new recombinant technology has been developed using baculovirus and butterfly pupa as a natural ‘bioreactor’ to produce highly purified ASF viral protein. The DNA code of a viral protein called P30 is inserted into the baculovirus DNA who is then injected into the butterfly pupa before being harvested. After purification, this P30 protein is used as reactive into a serological rapid-test. This is a new solution offered to the farmers to confirm the seropositivity of their animals directly at the farm without any equipment at a cost around 50% lower than ELISA. Rapid diagnostic tests were already available for antigen tests but this technology is the first to enable the production of rapid antibody tests.
Thanks to the purity of the P30 viral protein, the sensitivity of such technology is better than ELISA and should help to reduce the false-positive and reduce the cost of carcasses withdrawal for farmers.
With the increasing prevalence of the less virulent ASF strains, the reduction of mortality, and the increase of chronic forms of the disease, antibody tests are becoming more and more relevant. If you are interested in developing a better understanding of the nature of the antigen or antibody tests that you are using in your farms or your customers’ farms, I can connect you with our experts in that field.