​​Clinical, Pathological and Molecular Diagnosis of Peste des Petits Ruminants in Saudi Arabia

Peste des petits ruminants (PPRV) is a highly contagious disease among sheep and goats as well as wildlife ruminants. The virus has been reported and endemic in the Middle East, Africa and Asia (Balamurugan et al., 2010; Banyard et al., 2010; Alemu et al., 2019). The host affinity of the virus and different risk factors are responsible for the transmission of disease in different regions of the world (Kivaria et al., 2013; Chota et al., 2019; Herzog et al., 2020). The disease occurs with a higher incidence in the rainy season and the infection is mostly transmitted by close contact between animals (Lefevre and Diallo, 1990). It is of great economic importance based on mortalities, morbidity, loss of meat, milk and offspring (Murphy et al., 1999; Diallo, 2006). Singh et al., (2009) believed that the economic losses due to such disease may reach US$ 39 million/year. Nowadays, more than 1 billion goats and sheep in Asia and Africa are exposed to being infected with PPR (Bello et al., 2018). Therefore, the disease is considered one of the major threats to about 22 million small ruminants where mortality may reach 100%. Several studies indicated that outbreaks were more common and severe in goats (Balamurugan et al., 2012; Troung et al., 2014; Islam et al., 2021). The clinical signs of disease are pyrexia, anorexia, stomatitis, diarrhoea, occulonasal discharge and respiratory manifestations (Dhar et al., 2002; Muthuchelvan et al., 2006). The gross findings were congested lungs and gastrointestinal tract, especially the discontinuous streaks of congestion, which is referred to as Zebra stripes or Zebra markings, oedematous and congested retropharyngeal and mesenteric lymph nodes, linear hemorrhages in the intestinal mucosa (Ugochukwu et al., 2019).
       
Histopatholgy is characterised by pseudomembranous stomatitis and enteritis associated with bronchointerstitial pneumonia (Troung et al., 2014; Ugochukwu et al., 2019). A specific syncytial cells in oral mucosa and lung as well as intranuclear and intracytoplasmic inclusion bodies in respiratory and alimentary tract (Begum, 2021; Islam et al., 2021). PCR technique using F-gene primers has been developed (Forsyth and Barrett, 1995) and became a highly sensitive tool for diagnosis (Oshiek et al., 2018; Bhoyar et al., 2019). Phylogenetic studies indicate that PPR virus strains can be classified into 4 distinct lineages, three of them occur in Africa and the fourth is found in Africa and Asia (Shaila et al., 1996; Alidadi et al., 2019). In Saudi Arabia, Peste des Petits Ruminants (PPRV) is currently considered as one of the main transboundary viral diseases that constitute a threat to livestock production (Abu Elzein et al., 1990; Sharwai et al., 2010; Mahmoud et al., 2017; Mahmoud et al., 2021). Outbreaks are still repeated annually in the country and there is a big loss among all ages. Morbidity is up to 90% and mortality may reach 100% in young animals (Housawi et al., 2004; Al-Afaleq et al., 2004; Al-Dubaib, 2009; Boshra et al., 2015; Hemida et al., 2020). Therefore, the proper understanding of the epidemiology, pathology and molecular characterization of the strain of the virus in Saudi Arabia still needs further investigation. The present article is planned to study clinical signs, pathological descriptions and the molecular identification of PPR.

Animals
 
An outbreak among sheep and goats has appeared in winter 2021 (Al-Ahsa District). A total of 230 animals was examined (80 sheep and 150 goats) at Department of Pathology, King Faisal University, Saudi Arabia. These animals had not been vaccinated and there was also a history of the new entry of animals. The clinical signs were a sudden onset of fever with stomatitis and excessive salivation associated with mucopurulent discharges from the nose and eyes. Diarrhoea developed 3-4 days after the beginning of the fever. Mortality reached up to 90% particularly at young ages (< 1 year).
 
Postmortem and sample collection
 
Postmortem examination was carried out on dead animals. Tissue samples from the oral cavity, abomasum, small and large intestine, spleen and mesenteric lymph nodes, as well as lungs, were fixed in 10% neutral buffered formalin and processed routinely. Paraffin wax-embedded sections were stained with hematoxylin and eosin (HE) (Suvarna et al., 2019). Similarly tissues were also collected, transferred to the Central Biotechnology Laboratory at the College of Veterinary Medicine, King Faisal University, Saudi Arabia and stored at -80°C for PCR test.
 
RNA extraction
 
QIAamp Viral RNA Mini Kit (QIAGEN, USA) was used to extract total RNA according to the manufacturer’s recommendations. Briefly, 140 µl of the samples lysed by adding 560 µl of AVL buffer containing carrier RNA and then incubated at room temperature for 10 min. After complete lysis, absolute ethanol (560 µl) was added and mixed through pulse vortexing for 15 seconds. 630 µl Aliquots transferred sequentially to a QIAamp spin column followed by centrifugation at 8.000 rpm for 1 min. The binding RNAs were washed by AW1 (500 µl) buffer followed by AW2 buffer (500 µl). The RNAs eluted in 50µl of AVE buffer and stored at -80°C until used.
 
Molecular identification of PPRV
 
The presence of PPRV was tested in extracted RNAs using a One-step RT-PCR Kit (QIAGEN, USA). The RT-PCR reaction mixture was composed of 5 µl RNA, 5 µl 5x Qiagen one-step RT-PCR buffer, 5 µl Q buffer, 1 µl of a dNTPs mix, 1 µl (50 pmol) of each forward primer; 5'-CTTGATACTCC CCAGAGATTC-3' and Reverse primer; 5'-TTCTCCCATGAG CCGACTATGT-3' (Balamurugan, 2006), 1 µl of the enzyme mix (containing RT and PCR reaction enzymes) and 6 µl of RNase free water. The RT-PCR reaction was optimized at 50°C for 30 min, then 95°C for 15 min, followed by 40 cycles starting with denaturation at 95°C for 30 seconds, primers annealing at 57oC for 30 seconds and 72°C for 30 seconds and final extension step at 72°C for 10 min. The amplified PCR products were electrophoresed in 1.2% agarose gel containing 0.5µg/ml ethidium bromide and documented using an ultraviolet gel documentation system (BIORAD).

PPR virus is one of the major notifiable diseases of the world organization for animal health (OIE) and its existence can have a catastrophic impact on the region of farming development (Hemida et al., 2020). So far, the epidemiological situation of the virus in Saudi Arabia is not clear. During winter, 2021, The virus infects a large population of sheep and goats in different areas in Al Ahsa region. The animals were not previously vaccinated and also had a history of the new entry of animals. Previous studies stated that disease might be due to the change in the environmental conditions, illegal movement of animals through trade and husbandry practices (Kumar et al., 2014; Alemu et al., 2019). In the present study, PPRV was detected by RT-PCR in gel electrophoresis (Fig 1). The fragment size of the amplified products was 191 bp of M gene. This indicated the circulation of PPRV in the study districts. This finding was reported by previous studies (Balamurugan et al., 2012; Chitradevi et al., 2018; Shanmugavadivu et al., 2021). The clinical signs, necropsy findings and histopathology were quite similar in both sheep and goats. The clinical signs were sudden onset of fever with fibrinonecrotic stomatitis characterized by tiny, whitish, necrotic foci on the inner side of lips and gums stomatitis (Fig 2a). This was associated with excessive salivation and mucopurulent discharges from the nose and eyes. These findings were in agreement with previous studies (Truong et al., 2014; Islam et al., 2021). Diarrhoea developed 3-4 days after the beginning of the fever. The hindquarters soiled soft/water faeces and the perineal region was hyperemic and revealed whitish-grey necrotic foci (Fig 2b). Similar findings were recorded by several authors (Housawi et al., 2004; Hemida et al., 2020). The mortality rate was 90% particularly in young ages and these results were in accordance with previous studies (Mahmoud et al., 2017). The trachea, bronchi and bronchioles were filled with froth exudate.The lungs were congested and hepatized especially in the cranioventral lobes (Fig 2c). These findings were quite similar to those observed by several authors (Troung et al., 2014; Islam et al., 2021). The most common and constant findings in the intestine were severe enteritis as well as streaks of congestion in the colon and caecum (zebra stripes) (Fig 2d). These findings were very much consistent with several previous studies (Mahmoud et al., 2017; Robi, 2019; Ugochukwu et al., 2019). Histopathology revealed bronchointerstitial pneumonia characterized by thickening of alveolar septa (Fig 3a). The alveoli showed edema and alveolar macrophages. This finding was parallel to Troung et al., (2014) and Begum et al., (2021). In our findings, some syncytial cells appeared inside alveoli which were considered pathognomonic for PPRV (Fig 3b). This lesion was consistent with several previous studies (Kumar et al., 2014; Begum et al., 2021; Islam et al., 2021). Small and large intestine revealed fibrinonecrotic enterocolitis characterized by massive necrosis of mucosa accompanied by mononuclear infiltration as well as severe congestion of the submucosal blood vessels (Fig 3 c,d). This lesion was consistent with several previous studies (Housawi et al., 2004; Kumar et al., 2014; Troung et al., 2014; Islam et al., 2021).
 

 

 

The occurrence of the virus is considered an emerging pathogen in this district and affect susceptible host during adverse weather. Therefore, the state animal husbandry along with other stakeholders must keep a strict vigil on the development of disease among animals in susceptible areas. Also, quarantine of new animals before introduction to the herd and immediate implementation of mass vaccination, slaughter and proper disposal of affected animals is very important to prevent any further losses of disease.

This work was supported through the Annual Funding Track by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Project No. AN000312).

None.