Abstract: Foot and Mouth Disease (FMD) is one of the most contagious viral diseases of mammals that have an ability to cause high economic losses in susceptible cloven-hoofed animals. In addition to, losses in the milk productions occurred in the form of sudden and severe drop of milk yield. The aim of the present work was to throw light on the different methods for diagnosis and control of FMD that affect dairy small ruminants; sheep and goats. Sheep and goats play a role in the FMD epidemiology, as they become carriers and act as reservoirs of infection. Diagnosis of FMD achieved by many techniques such as virus isolation, Sandwich ELISA, Multiplex PCR, indirect ELISA (DIVA) and real time PCR. Virus isolation onto cell culture is considered as the “gold standard” technique for FMD diagnosis. Moreover, detecting of antibodies against the non-structural proteins (NSPs) of FMD using indirect-ELISA were successful for differentiation between infected and vaccinated animals (DIVA). Differentiation of the infected from the vaccinated animal is of great importance in the control program of FMD. The control program depends mainly on vaccination, treatment, effective quarantine measures, disinfection and hygiene and sanitation measures. Treatment protocols of small ruminants are showing typical clinical symptoms of FMD achieved by the use of antipyretic and analgesic medicine and a broad-spectrum long-acting antibiotic. The inactivated FMDV vaccine succeeded in reducing the outbreaks worldwide. It gives protection for all ruminants against FMDV for 1 year. Foot and mouth disease have the ability to cause milk production losses in small ruminants. Recent diagnostic tools urgently needed not only for the diagnosis, but also for following-up combating programs and control of FMD.
INTRODUCTION
Foot and Mouth Disease (FMD) is responsible for considerable economic losses through deaths in young animals or decrease in animal weight gain and milk production. The losses in milk production occurred due to severe and sudden drop in milk yield. Although, most adult livestock are able to recover clinically in 2-3 weeks, the re-establishment of the milk production level prior to FMD onset may require longer period resulting in severe economic losses1. Milk yield of different species and breeds of livestock studied before and after FMD infection. The FMD causes about 20 and 44% milk yield loss in cows and 19% losses in sheep. Moreover, FMD induced milk yield depression in Holstein, cross and local breed cows in rates of 37, 17 and 5%, respectively2,3.
Viruses represent serious threats to animal health. Consequently, early and quick diagnosis and identification of viral pathogens are essential. The diagnosis of viral diseases are important in the determining of the control strategies. In addition to the identification of the prevalence of viruses in different forms such as; serotypes or isolates are important. Many viral diseases such as; foot and mouth disease virus, peste des petits virus, bluetongue virus and infectious bovine rhinotracheitis and herpes viruses were continually inducing serious economic complications in the field4-8. Several molecular-based techniques like Polymerase Chain Reaction (PCR), probe hybridization9 and nucleic acid sequencing were widely used for this purpose10,11. Foot and mouth disease is one of the most contagious viral diseases of mammals that have the ability to cause high economic losses in susceptible cloven-hoofed animals12.
In Egypt, the FMDV outbreaks have been reported13 since 1950. Several foci detected during 1961-1970 and all infections were caused14 by strain O1, but in 2011, serotype A was isolated from Egyptian cattle in addition to the serotype15 O. In 2012, FMDV SAT2 reported in cattle and buffaloes in six outbreaks in 8 governorates in Egypt16. This FMDV serotype (SAT2) spread throughout Egypt, Libya and Palestine. Phylogenetic analysis of the isolated serotype (SAT2) showed that the circulating FMDV is genetically related to the isolated serotype from Saudi Arabia, Sudan, Eritrea and Cameroon10,17 between 2000 and 2010. A more recent outbreak belongs to the A/AFRICA/G-IV lineage occurred in Egypt, during 2016, the genetic investigation revealed its relationship to the collected16 viruses in Nigeria 2015 and Cameroon 2013.
Diagnosis of viral diseases depends on clinical signs, epidemiology, pathological lesions and specific detection of the viral antigen, viral genome or the specific antibodies in the tested samples by different serological tests and nucleic acid based assays18. The most common used diagnostic techniques are culture isolation, agar gel immunodiffusion (AGID), hemagglutination (HA) tests, immunocapture enzyme-linked immunosorbent assay (IC-ELISA) and competitive ELISA19. In addition to the previous techniques, Virus Neutralization Test (VNT) and reverse transcriptase polymerase chain reaction (RT-PCR) are used20,21. The control of FMD is not only necessary for reducing economic losses of the disease but also essential for increasing livestock production. The FMD control can also open up the new chance of export as it is a trans-boundary disease limit export of farm animal’s products from the country 22. The disease is endemic in Egypt as well as Africa, so the goal was to clarify the vision to reach a stage of control of the disease and declare those areas initially free of the disease with vaccination. Therefore, the aim of the current study was to throw light on the diagnosis and control of FMD that infect dairy small ruminants; sheep and goats.
DIAGNOSIS OF FOOT AND MOUTH DISEASE
Clinical picture: The diseased animals showed a prompt and severe lameness. So, the animals tend to set down frequently and have unwillingness to rise or move. Blisters found on the hoof, dental pad and sometimes tongue23,24. Small ruminants considered as the maintenance host for the FMD virus, not the propagative host like pigs. The clinical signs of FMD in sheep and goats are not severe like that of cattle and buffaloes and include high fever (41°C), salivation, decrease in food consumption and milk production, inability to move, oral vesicles, interdigital spaces ulcers, lesions on the dental pad and death of young animals (Fig. 1). In sheep and goats, the most frequent clinical sign is lameness. Affected animals develop fever, show reluctance to walk and may separate itself from the rest of the flock25. The incubation period of FMD in small ruminants is about 3-8 days. About 25% of the affected sheep do not develop vesicles and 20% have lesions only at one site or develop visible vesicles that last for few days. Vesicles may also observe on the teats especially of milking sheep and goats affecting milk yield and rarely on the vulva and prepuce26. Ewes may abort. unweaned lambs and kids usually die due to heart failure18,27.
Post-mortem examinations: The postmortem findings associated with FMD include vesicles and erosions of the mucous membranes of the mouth, rumen, teats and interdigital spaces, ulcers and lesions on the dental pad (Fig. 2)28.
| Fig. 1(a-c): | Different PM clinical signs of FMD |
| Source: DEFRA28 | |
Carrier status: Sheep and goats may also become virus carriers after exposure. Around 50% of recovering sheep insistently infected for up to 9 weeks and a small number of animals may carry the virus for up to 9 months. Some breeds of sheep can persist as carriers for up to 5 months after exposure. A number of mechanisms suggested for persistent infection with FMD virus.
| Fig. 2(a-b): | Different PM lesion of FMD |
| Source: DEFRA28 | |
The virus persists in the pharynx. Sheep and goats are less susceptible for FMD than cattle and pigs and the disease often has unapparent nature in these animals. Sheep and goats played a vital role in the FMD epidemiology as they become carriers and act as reservoirs of infection29. Animals could be carriers in the following cases; after recovery, sub-clinical form of FMD and when the vaccinated animals are subjected to the infection. All the three previous cases lead to the carrier’s state. The duration of the carrier state differs according to the species. Carrier state lasted for 5 years, 3 years and 9 months in African buffaloes, cattle and sheep and goats, respectively. Deer and antelope can convey the virus for long periods. Pigs do not become carriers30.
Differential diagnosis: Differential diagnosis of FMD applied to differentiate the diseases that misdiagnosed with it. These included the following; Peste des petit ruminants (PPR) can be excluded by signs of pneumonia and diarrhea, Bluetongue disease excluded by signs of facial oedema and nasal ulceration, Pock lesions exclude Capripox, Contagious ecthyma excluded by lack of vesicular stomatitis and lameness, Pneumonic Pasteurellosis and Caprine Pleuropneumonia are characterized by respiratory illness alone31.
Laboratory diagnosis
Virus isolation: According to the World Organization for Animal Health (OIE)32 , Terrestrial Manual, virus isolation onto cell culture is considered as the “gold standard” technique for FMD diagnosis33. This method is highly sensitive, but it is time-consuming, lasting between 1 and 4 days and requires extraordinary laboratory facilities. The most sensitive cell culture to most of FMDV serotypes is the primary bovine thyroid34, but they are difficult and exclusive and usually lose its susceptibility to FMDV after numerous passages35. Primary lamb kidney (LK) cells are very sensitive to FMDV and vary from primary bovine thyroid (BTY) cells in preserving of their sensitivity to FMDV infection after cryopreservation. Cell lines like baby hamster kidney (BHK-21) is much easier to preserve, but are less susceptible to specific animal-derived FMDV36. The summary of different assays for the diagnosis of FMD29 are given in Table 1.
Virus neutralization test: The virus neutralization test (VNT) is the “gold standard” technique for detection of antibodies to structural proteins of FMDV and is an approved test for the certification trade of animals and animal products37. The test sensitivity varies due to its dependence on various types of primary cells and cell lines cultures which have variable degrees of sensitivities to the FMD. In addition, VNT is time- consuming, liable for contamination and requires special facilities in comparison to other serological tests that can use inactivated viruses as antigens.
Nonstructural protein (NSP) antibody tests: Detecting of the antibodies against the non-structural proteins (NSPs) of FMDV successfully used for differentiation between infected and vaccinated animals (DIVA)38. Differentiation of the infected from the vaccinated animal is of great importance in the control program of FMD. This differentiation depends on the FMD vaccine quality that must be free from any live virus particles. A series of competitive and indirect ELISAs using 3AB3, 3ABC and truncated 2C (2 Ct) NSPs of FMDV was developed to achieve DIVA22,39. The 3ABC indirect ELISA routinely applied for general screening of bovines39. The sensitivity and specificity of the 3ABC indirect ELISA are 96 and 96.4%, respectively19. This assay has the ability to detect antibodies-3 ABC from 10-900 days post-infection in experimentally infected cattle. Recently, 3NSP based assays depending on 3B, 2B and 3D were developed and validated in India40,41.
Sandwich ELISA: Sandwich ELISA is rapid and simple to perform. It is the primary test for FMD diagnosis. The assay depends on serotype-specific polyclonal antibodies prepared in guinea pig and rabbits for the detection of FMDV structural proteins. The test gave 100% specificity and 80% sensitivity in FMDV detection29.
Complement fixation test (CFT): It is an old method in the history of clinical virology. The complement attacks antigen-antibody compound. Presence of the Ag-Ab complex triggers the complement to bind. Sensitized sheep red blood cells (RBCs) used as an indicative agent. Positive results are associated with no hemolysis. Although the CFT is convenient and requires low-cost materials, it is labor intensive and lacks sensitivity42.
| Table 1: | Different assays for the diagnosis of FMD |
| Source: Sharma et al.29 | |
Nucleic acid recognition methods
RT-PCR assay: Five FMDV serotypes were distinguished by the formerly published conventional RT-PCR techniques depending on the magnification of the VP1 gene43. Conventional RT-PCR techniques are serotyping specific. Consequently, conventional RT-PCR recommended just for amplification of the VP1 region due to its incompatibility to serotyping followed by further nucleotide sequence analysis10,37.
RT-LAMP: It is an isothermal nucleic acid amplification technique, which carried out at a constant temperature and does not require a cycler like PCR44. RT-LAMP is extremely sensitive molecular analyses for the simple and rapid detection of FMDV. A total of 38/50 samples were positive by RT-LAMP and the identified serotypes were A (15/50), O (15/50) and Asia-1 (8/50)45. The RT-LAMP succeeded in the amplification of 199, 209 and 187 bp of the target sequence of the 3D polymerase gene of serotypes A, O and Asia-1 at 60°C during 15-60 min, respectively. A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) developed using general and serotype-specific genes in a single tube. This test easily performed and can detect FMDV at serotype level in about 60 min. In addition, it has a comparable sensitivity and specificity to reverse transcriptase PCR and real-time PCR46.
Multiplex PCR: The technique is more rapid and sensitive than conventional virus isolation47. Assays were established and directed against the conserved 3D region and 5' UTR region of the FMD virus. Afterward, the multiplex PCR applied for detect FMDV (mPCR) directed to the VP1 region was developed and differentiated the serotypes36. In this assay, 2 primers sets were used, the first targeting the 1D region and the second direct to 2 B region. The technique succeeded in the identification of FMD serotype. Products of different sizes (249, 376 and 537 bp) were obtained which are specific for serotypes O, A and Asia1, respectively. The minimum detection limit of the mPCR has been valued10,48 as 1×10–1 TCID50/mL for serotypes O, A and Asia 1.
Real-time RT-PCR: These assays settled for the identification of FMDV all over the world. A PCR assay directed to the 1D region established in a multiplex design for concurrent detection and identification of FMDV serotypes in the samples49. The RT-PCR assay was very sensitive, because of the great sensitivity and specificity of the RT-PCR assay, it was suggested as the main test for the FMDV detection in persistently carriers. This technique is of extreme significance in disease control as it can detect the carrier animals50.
CONTROL OF FOOT AND MOUTH DISEASE
Quarantine measures and disinfectants: It is necessary to apply thorough cleaning and proper disinfection to all premises and infected materials, such as implements, cars and clothes. Hygienic removal of carcasses, bedding and contaminated animal products are important32. In free areas like Britain, USA and Sweden applied extensive program for FMDV control that depends on stamping-out; killing and destruction of all infected animals and their immediate susceptible contacts followed by thorough cleaning and disinfection of the affected premises24,51. The FMDV is liable for wide variety of disinfectants such as; sodium hydroxide, carbonate, citric acid and Virkon-S26. The FMD virus is defenseless to excesses of pH. Therefore, both acids (e.g., citric acid) and bases (e.g., caustic soda or sodium hydroxide) have the ability to destroy the virus. There are many marketable types of disinfectants that be used in the elimination of the FMD virus. Strict precautions and proper usage of the exact concentration of the disinfectant according to the manufacturer instructions recommended52.
Treatment of FMD in small ruminants: Treatment protocols of small ruminants are showing typical clinical symptoms of FMD achieved by the use of antipyretic and analgesic medicine (Vetalgin-Intervet) and a broad-spectrum long-acting antibiotic (Terramycine/LA Pfizer) and both protective dressing or immunomodulatory was applied53. Localized treatment by rinse the ulcerative vesicles found on the mouth, tongue, legs, claws and teat with one of the following solutions; normal saline, citric acid 1% potassium permanganate 1% or alum 2% were helpful54.
FMD VACCINES
Attenuated vaccines: Attenuated strains produced by the passage in the laboratory animals like mice and rabbits or in the embryonated eggs until their virulence for infection were weak or lost55. This vaccine has some disadvantages, it does not allow discrimination of naturally infected and vaccinated animals, attenuated vaccine virus spreading to non-vaccinated livestock and may cause the development of virus carriers56. There many types of FMD attenuated vaccines such as; Novel Attenuated Vaccines, Nucleic Acid Vaccines, Synthetic Peptide Vaccines, Viral Capsids Vaccines, Virus-Like Particles Vaccines (VLP) and Nanoparticle Vaccines, but all of them are under research or not authorized.
| Table 2: | The FMD vaccines in Egypt |
| Source: CFSPH26 | |
Inactivated killed vaccine: The vaccine produced from live FMDV amplified in BHK-21, then inactivated by special chemicals like formalin, purified and mixed with the adjuvant. It is of great importance to insure the freedom of the vaccine from any live virus particle to avoid post vaccinal infection and allow the DIVA51. The inactivated FMDV vaccine succeeded in reducing the outbreaks worldwide. There are a number of limitations with its use in emergency control programs; one of these limits is that the incomplete inactivation may lead to infective vaccines57. The inactivated vaccine gives protection for all ruminants against FMDV for one year. The first dose for cattle and buffaloes is 3 mL and for sheep and goats is 1.5 mL. A booster dose injected subcutaneously 3-4 weeks after the first dose. The dairy cattle, breeding calves, sheep and goats vaccinated at 6-8 months age58. The details of some FMD vaccines is given in Table 226.
CONCLUSION
Foot and mouth disease (FMD) is one of the most contagious viral diseases of mammals that have the ability to cause high economic losses in susceptible cloven-hoofed animals. The most frequent clinical signs associated with FMD in sheep and goats are lameness, high fever (41°C), salivation, oral vesicles, interdigital spaces ulcers, lesions on the dental pad, severe drop in milk yield in dairy animals and death of young animals. Diagnosis of FMD achieved by many techniques such as; virus isolation, Sandwich ELISA, Multiplex PCR, indirect ELISA (DIVA) and real time PCR. The control program of FMD depends mainly on vaccination, effective quarantine measures and hygiene and sanitation measures. The inactivated FMDV vaccine succeeded in reducing the outbreaks worldwide. It gives protection for all ruminants against FMDV for one year.
SIGNIFICANCE STATEMENT
Foot and mouth disease is one of the most contagious viral diseases that have the ability to cause high economic losses in susceptible cloven-hoofed animals. It causes severe drop in milk yield in the dairy small ruminants. Early and accurate diagnosis is necessary and the recent diagnostic tools urgently needed not only for the diagnosis but also for following-up combating programs and control of FMD. This study found that the inactivated FMDV vaccine succeeded in reducing the outbreaks and recommended in endemic areas. Moreover, this study discussed the different methods used in diagnosis and control of FMD that will help the researchers in exploring and discovering the methods suitable in their countries.