Clinical and diagnostic study of ovine chronic progressive pneumonia (Maedi-Visna) in sheep of Basrah, Iraq

1. Clinical study of chronic progressive pneumonia (Maedi-Visna) in sheep of Basrah, Iraq.
2. Hematological evaluation of diseased sheep.
3. Macroscopic and microscopic investigation of dead carcasses affected with the disease.
4. Serological diagnosis of diseased animals using ELISA test.

Introduction

Ovine chronic progressive pneumonia (Maedi-visna) is a persistent virus infection distributed globally and considered an essential chronic viral disease of sheep and goats with a high worldwide prevalence. It is caused by a Lentivirus with characteristic clinical signs including chronic pneumonia, mastitis, encephalitis and arthritis, which may also be observed in diseased animals (1-3). The disease causes significant economic losses because of animal culling or high mortalities of diseased animals and depressing animal productivity (4). The disease is characterized by the lifelong persistence of the causative virus in host monocytes and macrophages. However, a changeable prolonged time between the onset of the disease and the stimulation of a serologically detectable antiviral antibody response might be needed (5). The disease could infect sheep and goats even though, diseased animals do not show obvious signs, However, they stay persistently infected and can transfer the causative agent (6,7). Maedi-visna refers to labored or exhausted breathing (Maedi), which reflects progressive interstitial pneumonitis. Moreover, wasting or shrinkage (Visna) is another clinical manifestation diseased animals, reflecting paralyzing meningoencephalitis. On the other hand, progressive pulmonary disease is the primary finding with Maedi-visna virus infection (2,8). It was shown that the high speed of the disease spreading due to flaws or lateness of treatment or vaccination programs and the lack of prevention measurement plan protocols, resulted in control difficulties, as did the lack of active protocols for early and fit diagnosis of diseased patients using precise and specific serological and molecular techniques (9,10). Furthermore, isolation and characterization of Maedi-visna infection might not always be attempted for usual diagnostic procedures because of the persistent nature of the virus. Therefore, stability and a positive antibody status could be enough to identify and detect the carrier status. Furthermore, because of the seroconversion, which comes late after the viral infection, a negative serological status might indicate recently infected animals (11,12). It was documented; that the World Organization for Animal Health (OIE) included Maedi-visna disease the list of notifiable contagious diseases that considerably affect animal world trade and products. Moreover, in ovine, the economic losses related to Maedi-visna infection could be more evident and effective (1). It was shown that ELISA is the common screening test used for detection and surveillance. It detects antiviral antibodies with high sensitivity and specificity regarding disease occurrence and seroconversion (13,14). Notwithstanding, serum-positive samples do not indicate or approve the clinical disease. On the contrary, a seronegative animal also cannot be considered disease-free. Consequently, seroconversion might require a period even at the beginning of the infection. The titer antibodies could reflect the differences within an animal's life. However, in some diseased cases, antibodies could not be detectable using different ELISA tests (depending on the antibody titration and the type of ELISA applied), which may indicate the inaccuracy of this test for an accurate and final diagnosis (15,16).

Ovine chronic progressive pneumonia (Maedi-visna) is suspected in sheep of Basrah, Iraq, for the first time causing high morbidity and mortality rates, Therefore, clinical and diagnostic studies were applied.

Materials and methods

Ethical approve

The Animal Ethics Committee at the College of Veterinary Medicine, University of Basrah, Iraq, allowed this research to be conducted in accordance with the official letter No. 7/18/4729 dated 2023.

Study design

The study assessed 135 local sheep breeds 3-5 years old and of both sexes reared in Basrah province, Iraq. The study started from October 2023 to June 2024. Suspected animals show signs of chronic pneumonia, inappetence, emaciation, progressive wasting, and chronic cough. Twenty-five clinically healthy local sheep breeds were served as a control group. Diseased and control groups were subjected to complete clinical and clinical pathological examinations to exclude common clinical diseases such as blood parasitic, viral, gastrointestinal parasitic infection and nutritional diseases using the usual laboratory procedures.

Sampling and hematological assessment

Fifteen milliliters of blood were withdrawn from the animal jugular vein from each diseased and control animal, From these, three milliliters (3) of blood were mixed with EDTA for evaluation of the complete blood picture, including, the total erythrocyte count (RBC), hemoglobin concentration (Hb), packed cell volume (PCV), total thrombocytes count, thrombocytes distribution with, the mean thrombocytes volume and total leukocytes count (TLC) using a general blood analysis device from (GENEX . USA). Moreover, differential leukocyte count was applied by using blood smears according to the Giemsa stain technique (17). On the other hand, three milliliters (3) of blood were mixed with Trisodium citrate to estimate blood clotting factor indices (using plasma) (prothrombin time, activated partial thromboplastin time, and fibrinogen time), using special kits from Biolabo, France. Furthermore, the erythrocyte sedimentation rate (ESR) was also estimated using the Wintrobe tube technique, according to Weiss and Wardrop (18).

Macroscopic and histopathological examinations

Forty dead carcasses confirmed with ovine chronic progressive pneumonia were subjected to postmortem macroscopic and histopathological examinations and evaluation. Hereby, according to Cullen, Stalker (19). Samples were extracted from the lungs of dead animals and placed in a 10 percent formaldehyde solution (buffered neutral). Specimens were cleaned with tap water following seventy-two hours of fixation. After that, regular handling and processing were performed with a series of alcohol concentrations starting from 70 to 100% absolute alcohol for two hours at each concentration to remove water from the processed tissues. Moreover, xylol was used during the clearing procedure. Afterward, specimens were embedded using liquid paraffin wax at 58°C in two phases to create specimen blocks, which were sectioned at a thickness of 5 mm for all tissues. Finally, sectioned tissues were stained with hematoxylin and eosin stain (19).

Indirect ELISA test

To investigate the specific IgG immunoglobulin, an ELISA assay for Maedi-visna was applied according to the manufacturer’s procedures from CREATIVE DIAGNOSTICA (USA.

Statistical analysis

SPSS (student t-test), according to Leech et al. (20). Statistical calculations were used in the current work to estimate and calculate the statistical difference between the diseased and control groups. The significant value was set at (P<0.05).

Results

Clinical and Hematological results data

Results indicated that out of 135 sheep serum examined via the indirect ELISA test 88 65.18% was positive for the specific IgG of Lentivirus casing ovine chronic progressive pneumonia Maedi-visna. Where, infected animals exhibited various clinical signs, including loss of appetite which was seen in 81.8% of diseased sheep, dyspnea with an obvious increase in abdominal breathing 78.4%, chronic cough with abnormal lung sound (dry rales and/or crepitation) 73.8%, an extension of the neck with nostrils expansion 73.8%, progressive weakness and emaciation 72.7%, decrease milk production in lactating ewes 37.5% and lameness 31.8% (Table 1). On the other hand, during the clinical examinations, diseased sheep showed average body temperature associated with a significant rise in respiratory and heart rate compared with the control group (Table 2). Hematological analysis of diseased sheep affected with Media/visna compared with control healthy sheep indicates a significant increase in ESR values, A significant leukocytopenia due to a decrease in total leukocyte count and a significant lymphocytopenia, as well as monocytopenia in diseased sheep, compared with the control group (Table 3). Concerning the indices of clotting factor of the infected sheep group and the controls, the results indicated a significant decline in total thrombocyte count in the diseased sheep group than in the control group. While a significant increase has been encountered in the amount of the distribution width of the platelets, the mean volume of the platelets, prothrombin time, the activated partial thromboplastin time, as well as the fibrinogen time of the diseased sheep group compared with the control group (Table 4).

Table 1: Clinical manifestations of sheep suffering from Maedi-visna

Table:2: Body temperature, Respiratory, and heart rate of diseased sheep with Maedi-visna and control group

Mean±Standard error of the mean. * Significant at P<0.05.

Table 3: Hematological changes of diseased sheep with Media-visna and control group

Mean±Standard error of the mean. * Significant at P<0.05.

Table 4: Changes in the indices of the clotting factor of sheep with Maedi-visna and the control group

Mean±Standard error of the mean. * Significant at P<0.05.

Postmortem and histopathological examination

Postmortem examinations confirmed that dead carcasses with ovine chronic progressive pneumonia (Maedi-visna) show signs of progressive pneumonia, whereby the affected lung was enlarged and heavy without any collapse. Moreover, there are apparent rib impressions on the costal surface of the affected pulmonary lobe. In addition, the lung has patchy hemorrhagic lesions (Figure 1). On the other hand, diseased lungs also show variable nodular sizes with whitish nodules on the parietal surface. Moreover, the affected lung failed to collapse. Further, variable whitish patches on the parietal surface of the affected lung were also detected (Figures 2 and 3). Nonetheless, the histopathological appearance of dead sheep with Maedi-visna shows lobar pneumonia consists of progressive inflammatory exudation filling the lobar alveolar spaces with severe infiltration of inflammatory cells, and erythrocytes inside alveolar spaces, referring to the congestion stage of pneumonia. Furthermore, progressive inflammatory exudation filling the lobar alveolar spaces of the lung consists of inflammatory cell infiltration and exudation with erythrocytes inside the alveolar spaces. Also, a hyperplastic proliferation of the bronchial epithelial lining is indicated in diseased lungs (Figures 4 and 5). On the other hand, early fiberoptic lesion refers to the stage of red hepatization of pneumonia and many inflammatory cells and erythrocytes infiltration in the alveolar spaces (Figure 6).

Figure 1: The gross appearance of the lung shows progressive pneumonia, whereby the affected lung is enlarged and heavy without any collapse. Moreover, there are obvious rib impressions on the costal surface of the affected pulmonary lobe (black arrow), In addition, there are patchy hemorrhagic lesions on the lung (blue arrow).

Figure 2: The gross appearance of the lung shows progressive pneumonia, The affected lung is enlarged and has variable modular sizes with whitish nodules on the parietal surface, in addition, the affected lung failed to collapse (black arrow), and there are diffuse hemorrhagic patches on the lung (blue arrow).

Figure 3: The gross appearance of the lung shows progressive pneumonia, The affected lung is enlarged and has variable whitish patches on the parietal surface, Moreover, the affected lung failed to collapse (black arrow); there are diffuse hemorrhagic patches on the lung (blue arrow).

Figure 4: This histopathological micrograph of lobar pneumonia shows progressive inflammatory exudation filling the lobar alveolar spaces (black arrows), severe infiltration of inflammatory cells, and RBCs inside alveolar spaces (blue arrows). referring to the congestion stage of pneumonia. The H&E stain, is 10X.

Figure 5: Histopathological micrograph of progressive inflammatory exudation filling the lobar alveolar spaces of the lung (black arrows) consists of inflammatory cell infiltration and exudation with RBCs inside the alveolar spaces, Also there is a hyperplastic proliferation of bronchial epithelial lining (blue arrows). H&E stain, 40X.

Figure 6: Histopathological micrograph of the lung shows an early fiberoptic lesion referring to the stage of red hepatization of pneumonia (black arrows) as well as many inflammatory cells and RBCs infiltration in the alveolar spaces (blue arrows). H&E stain, 40X.

Discussion

Scientific workers proved and indicated that ovine chronic progressive pneumonia (Maedi-visna) in sheep is caused by the Maedi-visna virus, a small lentivirus that causes a chronic type of infection and different inflammatory lesions in diseased sheep and goats. Pneumonia and mastitis are the most common clinical features. Moreover, the virus's most target tissues are pulmonary, mammary, and nervous. However, joints could also be affected (4,21,22).

It was shown that the clinical appearance of Maedi-visna could depend on the virulence and strain of the causative agent, the reflected immune response of the infected host, and the susceptibility of the host, including the genetic resistance to the causative virus (23). Moreover, it was documented that, the predominance of infected cases, slow virus replication, and the considerable value of infected erythrocytes in the bloodstream are minimal (2). Hereby, Maedi-visna turns to latent form or progressive. Therefore, diseased animals could not show clear or obvious signs of the disease, particularly at its early stages (2,24). On the other hand, any suppression of immunity due to senility (old age), the presence of concurrent diseases, and the stress of the environment might increase and enhance the replication of the virus, Therefore, clinical manifestations of the disease become more obvious and visible (25). In the current study, diseased sheep exhibited different clinical manifestations, which were also mentioned by Constable et al. (2), Minguijón et al. (3), and Gomez-Lucia et al. (24). Chronic respiratory manifestations are the more common clinical feature of the disease (21).

It was shown that the most important clinical manifestations resulting from Lentivirus infection in sheep and goats are progressive pneumonia, meningoencephalitis, and synovitis with obvious chronic arthritis. However, some of those manifestations could only occur in newborn animals (26,27). Moreover, Luqman and Cumali, (15) and Brinkhof et al. (28) documented that a high prevalence of infection was recognized in sheep showing respiratory symptoms, or what they call (breathing ewes) compared with clinically normal sheep. On the other hand, Constable et al. (2) and Gomez-Lucia et al. (24) also agreed that the first targeting of Maedi-visna virus in animals was in the respiratory system, which developed into more severe respiratory distress and was associated with a progressive wasting type, as indicated in the current study. Furthermore, Blacklaws (29) describes the relationship between respiratory and other clinical manifestations and disease seropositivity.

In the present work, the final confirmation of the diagnosis of the disease was proved by the detection of the virus antibodies in sheep serum using an indirect ELISA test, whereby it was shown that the multiplication of the Lentivirus could be detected in more restricted cells reflecting more restricted antigen productions by the immune system and then might reflect an obvious immunological reaction (13). On the other hand, it was documented that various ELISA tests can be used to confirm the causative virus antigens in diseased animals' serum. However, most of those ELISA are indirect types of ELISA. Nevertheless, a competitive ELISA test using monoclonal antibodies also showed high sensitivity and specificity for detecting of the virus antigens (24,29,30). It was also mentioned that the ELISA test was used globally to confirm the diagnosis and apply the control and eradication plans for the disease in sheep and goats (31).

Al-Baroodi et al. (13) and Brinkhof and Van Maanen (28) indicate that early and inevitable diagnosis of the disease is a definitive criterion for controlling and eradicating of the disease. as any control and eradication programs should be based on serological tests, mainly the ELISA test to detect the specific antibodies against the virus. At this moment, only the seropositive animals are considered infected and subsequently removed from the flock, which can be considered the main snag for eradication of the disease. Whereby the immune response of Maedi-visna needed a longer period (14). The hematological changes in the present work indicated significant leukocytopenia, lymphocytopenia and monocytopenia, Zhang et al. (32) and Pérez et al. (33) also indicated the same results.

 The most important incident that assists viral replication has yet to be fully know. However, the immune response created in the diseased animal's body reflected a chronic inflammation causing different pathological changes, including infiltration of mononuclear cells, such as lymphocytes, and macrophages, as well as the plasma cells which will be arranged into forms like the lymphoid follicles and could devastate the normal tissue structure and affected tissue functions (2,4). Moreover, increased levels of Granulocyte-macrophage colony-stimulating factor in Maedi-visna may contribute to developing of lymphoid follicles in the lungs (chronic lymphocytic interstitial pneumonia) in animals infected with Maedi-visna. However, it is unclear whether the increased expression of the Granulocyte-macrophage colony-stimulating factor in vivo is the direct consequence of the causative virus (32).

Leukocytopenia is a clear decrease in the circulating total white blood cells, which might result from depression of their production, increased utilization destruction or both (17). On the other hand, lymphocytopenia reflects a decreased number of lymphocytes in the blood, has a vital role in immune protection (18). It was documented that viral infection is mostly characterized by lymphocytopenia due to decreased re-circulation of lymphocytes from lymphoid tissues. However, stress arising from chronic diseases plays a good role too (17). Moreover, Constable et al. (2). added that the causative Lentivirus infects the monocytes (the macrophage lineage) and adheres to those cells by linking its glycoprotein envelope to specific receptors on the cell surface, which results in cell disintegrations and depletions that reflect monocytopenia in most diseased cases. Furthermore, the production of viral antigens attracts more monocytes.

Results also indicate a significant increase in ESR in diseased sheep than in the control group. The rate of erythrocyte sedimentation readings frequently increases in diseased animals with anemia or tissue damage compared to normal healthy sheep. Hereby, the highly elevated values of ESR found in the current study of infected sheep may reflect the obvious inflammatory reaction induced by Maedi-visna, especially in the pulmonary tissue of infected sheep. Moreover, it was also documented that, increased fibrinogen levels (hyperfibrinogenemia) when indicated in the blood will also play an important role in causing erythrocytes to clump, and the sedimentation rate will be enhanced rapidly (18,33).

In the current study, the results indicate a significant difference in the clotting factor indices induced by the Maedi-visna virus in diseased sheep, which could reflect the occurrence of a disseminated intravascular coagulation syndrome and a bleeding tendency of infected animals seen in tissues of dead carcasses, whereby the intensity of this coagulopathy depends on the severity and duration as well as the ischemic and inflammatory problems indicated by the disease (34). It was believed that hypofibrinogenemia and decreased thrombocyte count, with less clotting time, could reflect the appearance of hemorrhagic patches indicated on different body tissues, especially on the lungs, which may be explained by the release of some endogenous mediators such as platelet-activating factor reflected by the inflammatory disorders (35).

Most scientific reports on Maedi-visna refer to different macro and microscopic pathological changes detected in dead animals, which is also indicated by the current study, where they believe that lesions in different tissues always arise from various immune reflexes induced via the antigen (Maedi-visna virus) as well as the replication of the virus itself (2). Although it was mentioned, most tissues could be affected by the causative virus, including lung, mammary, and nervous tissues. Nonetheless, the current study indicated that the lesions in both lungs of dead sheep were affected by the disease (26).

Luján et al. (36) mention that the primary macroscopic lesion is an enlargement of both lungs with increased weight. Furthermore, Maedi-visna could also affect regional lymph nodes (24). Moreover, chronically affected lungs will be hypertrophic. They might be up to three times their normal size, with grayish yellow, rubbery, rounded, and focal to diffuse red and/or gray spots that could be seen on the pulmonary pleural surface. Furthermore, an enlargement of mediastinal lymph nodes could also be indicated (2).

 It was indicated that the histopathological changes seen in pulmonary tissues of Maedi-visna diseased sheep reflect a characteristic chronic interstitial pneumonia with the proliferation of lymphoid tissue and numerous lymphoid follicles (36). On the other hand, Constable et al. (2) added that any secondary microbial infections will lead to engorgement and redness of the cranioventral area, which might become moist with the marked lobular pattern. Further, it was also documented that the histopathological feature of diseased lungs indicates an increase in the thickness of the alveolar septa, resulting in a decreased air exchange capacity of affected lungs (37-40). Moreover, the intra-alveolar septa are infiltrated with a large number of monocytes, lymphocytes, and macrophages, as well as plasma cells, which enhance increased thicknesses and hyperplasia of the smooth muscle fibers of the alveolar walls reflected pulmonary dysfunction and severe respiratory distresses (2). On the other hand, red patches could indicate clotting factor disturbances enhancing the blessing tendency process (24). Further, Luján et al. (36) added that diffuse lymphoid hyperplasia, detected in the histopathological view of diseased lungs might reflect and be responsible for the gray spots sometimes indicated in the pleural membrane. As the disease develops, the pulmonary lesions become fibrotic (which is indicated in the current study), reflecting an overgrowth, hardening, and scarring due mostly to a reparative response to damage and is attributed to excess deposition of extracellular matrix components, including collagen (37-45).

Conclusions

The current study concluded that Maedi-visna considered one of the chronic progressive diseases that harm farm animals, ended mostly with high mortalities. Therefore, the haste to take preventive measures to limit its spread must always be considered.

Acknowledgments

The authors extend their thanks and gratitude to the College of Veterinary Medicine, University of Basra, Iraq.

Conflict of interest

In this article, there are no conflicts of interest.