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Pathophysiology of peste des petits ruminants in sheep (Dorper & Kajli) and goats (Boer & Beetal)
Accepted Manuscript Pathophysiology of peste des petits ruminants in sheep (Dorper & Kajli) and goats (Boer & Beetal) Ahrar Khan, M. Kashif Saleemi, Farah Ali, Muhammad Abubakar, Riaz Hussain, Rao Zahid Abbas, Imtiaz Ahmad Khan PII:
S0882-4010(18)30008-1
DOI:
10.1016/j.micpath.2018.02.009
Reference:
YMPAT 2775
To appear in:
Microbial Pathogenesis
Received Date: 2 January 2018 Revised Date:
4 February 2018
Accepted Date: 6 February 2018
Please cite this article as: Khan A, Saleemi MK, Ali F, Abubakar M, Hussain R, Abbas RZ, Khan IA, Pathophysiology of peste des petits ruminants in sheep (Dorper & Kajli) and goats (Boer & Beetal), Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.02.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Pathophysiology of Peste Des Petits Ruminants in Sheep (Dorper & Kajli) and
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Goats (Boer & Beetal)
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Ahrar Khan1*, M. Kashif Saleemi1, Farah Ali2, Muhammad Abubakar3, Riaz
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Hussain2, Rao Zahid Abbas1 and Imtiaz Ahmad Khan4
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Pakistan.
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Faculty of Veterinary Science, University of Agriculture, Faisalabad-38000,
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Bahawalpur- 63100, Pakistan.
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University College of Veterinary and Animal Sciences, The Islamia University of
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National Veterinary Laboratory, Islamabad, Pakistan
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Department of Pathobiology, PMAS-Arid Agriculture University, Rawalpindi-
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46000, Pakistan
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17 Corresponding Author:
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Ahrar Khan (
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Department of Pathology,
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Faculty of Veterinary Science,
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University of Agriculture,
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Faisalabad-38000, Pakistan;
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[email protected]; [email protected]
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Phone: +92 333 651 7844
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https://orcid.org/0000-0001-5492-426)
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Page 1 of 23
ACCEPTED MANUSCRIPT Abstract
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Peste des petits ruminants (PPR), an economically important viral transboundary
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disease of small ruminants is not only prevalent in Pakistan but also in other countries
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where people rely on agriculture and animal products. The present study was aimed at
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describing the pathology and antigen localization in natural PPR infections in local
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(Kajli sheep; Beetal goats) as well as imported small ruminant breeds (Dorper sheep;
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Australian Boer goat). Morbidity and mortality rates were significantly (P < 0.001)
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higher in indigenous Kajli sheep (75.37 and 32.80 %) and Beetal goats (81.10 and
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37.24 %) as compared to Dorper sheep (6.99 and 1.48 %) and Australian Boer goat
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(5.01 and 2.23 %). Affected animals exhibited high fever, severe diarrhea, abdominal
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pain, respiratory distress and nodular lesions on lips and nostrils. Thick mucous
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discharge was oozing out from nostrils. On postmortem examination, lungs were
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congested and pneumonic, with nodular and cystic appearance and intestines were
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hemorrhagic with zebra stripping. Characteristic histopathological lesions of PPR
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were noted in intestines, lymphoid organs and lungs. In GI tract, stunting and blunting
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of villi, necrotic enteritis, and infiltration of mononuclear cells in duodenum, jejunum
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and ileum. Small intestines exhibited diffuse edema of the submucosa along with
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proliferation of fibrocytes, leading to thickened submucosa which has not been
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reported previously. Lymphoid organs showed partial to complete destruction of
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lymphoid follicles. Lesions of the respiratory tract included depictive of
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bronchopneumonia, severe congestion of trachea and apical lobe of lungs with
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deposition of fibrinous materials. Histopathological lesions of respiratory tract were
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severe and characteristic of broncho interstitial pneumonia, bronchopneumonia,
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interstitial pneumonia and fibrinous pneumonia. The alveoli were filled with
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edematous fluid mixed with fibrinous exudate, numerous alveolar macrophages,
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mononuclear cells along with thickened interalveolar septa and presence of
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intranuclear eosinophilic inclusions. One-Step RT-PCR using NP3 and NP4 primers
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confirmed a PPR virus of 352 bp size in spleen, lungs and mesenteric and brachial
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lymph node samples. It was concluded that morbidity and mortality due to PPR were
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significantly higher in indigenous breeds of sheep and goat as compared to imported
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sheep and goat breeds. PPR has rendered various lesions in GI and respiratory tract
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which are characteristic in nature for the diagnosis of the disease under field
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condition.
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Keywords: Peste des petits ruminants, sheep, goat, morbidity, mortality, gross and
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histopathological lesions of GI and respiratory tract
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1.
Introduction
66 The economy of Pakistan is agro-livestock based and role of livestock in agriculture
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sector is pivotal in rural socio-economic development [1-3]. Nearly eight million
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families in the country are involved in livestock raising. This sector is a source of
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income, and often the only source of income, for the rural and most marginal people,
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thus playing a key role in poverty alleviation [4,5]. Livestock sector is contributing
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approximately 58.6 % to the agriculture value added and 11.6 % to the overall GDP in
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Pakistan. Sheep and goats, being dual purpose animals with estimated population of
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70.3 and 29.8 million heads, respectively, are used for meat and to some extent for
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milk production [6].
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Sheep and goat farming in Pakistan is progressing rapidly and is contributing
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significantly to the national economy. However, its growth is being hampered by
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various diseases [7], including respiratory ailments like peste des petits ruminants
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[8,9]. Peste des petits ruminants (PPR) is reportable to the World Organization for
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Animal Health (OIE) and is an economically important disease in parts of Africa and
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some Asian countries. It is among transboundary diseases [10,11] and is not only
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present in Pakistan [12-15] but also in surrounding countries, including Afghanistan
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[16,17], India [18-21], Iran [22] and China [23,24]. This disease has also been
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reported from east African and Middle East countries and from most third world
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countries where people rely on agriculture and animal products [25,26].
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PPR is primarily a disease of sheep and goat but it has also been reported in
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Ethiopian camels, Indian buffalos [27], Saudian gazelles and deers [28] etc. Peste des
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petits ruminants, also known as goat plague and ovine rinderpest, affects sheep, goats
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and related species of small ruminants [29] and is highly contagious viral disease
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[30,31]. Morbillivirus, the causative agent, is closely related to rinderpest, measles
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and canine distemper virus [17,32]. The principal pathological findings of PPR are
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seen in the digestive and respiratory systems [33,34]. Morbidity and mortality could
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be 5-90 and 50-80 %, respectively [35,36].
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Vaccination is a strategy to control PPR and the vaccine is required at mass
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scale, however, losses can be curtailed through preventive measures for which Page 3 of 23
ACCEPTED MANUSCRIPT knowledge of pathology and pathogenesis of the disease is of vital. Studies of PPR
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usually revolve around sero-epidemiology [12,36,37] and rare efforts have been made
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to report its pathology. Most of the available information on the pathology of PPR has
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been based on results obtained following experimental infections [38] but none in
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Dorper or Kajli sheep and Boer or Beetal goats under field conditions. Data regarding
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pathological parameters from field cases are very important as these can better
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characterize the pathogenicity of the circulating virus. However, such data of PPR in
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Dorper/Kajli sheep and Boer/Beetal goats are lacking in the published literature. The
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severity of the disease varies with species, immunity and breed of the host [29]. The
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present study was aimed at describing the pathology and antigen localization in
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natural PPR infections in exotic sheep and goat breeds (Boer goat and Dorpar sheep)
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kept in close contact with local sheep (Kajli) and goat (Beetal) breeds.
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2.
Materials and methods
2.1.
Study Area and Environment
Lahore is the capital city of the Punjab province of Pakistan. It is the second most populous city in Pakistan and the 32nd most populous city in the world [39]. The
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city is located in the north-eastern end of the province, bordering with the Indian state
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of Punjab. Lahore has a semi-arid climate. Lying between 31°15′ - 31°45′ N and
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74°01′ - 74°39′ E, Lahore is bounded on the north and west by the Sheikhupura
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District, on the east by Wagah, and on the south by Kasur District. Study area was in
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suburban of Lahore towards Kasur District. Here, the hottest month of the year is
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June, with an average temperature of 40 °C (104.0 °F) and the coldest month is
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January at 14°C (56°F) with dense fog [1]. The monsoon season starts in late June,
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and the hot and humid month is July with heavy rainfalls, high humidity (81 %) and
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evening thunderstorms with the possibility of cloudbursts. This study was conducted
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in suburban of Lahore towards Kasur District during the mid of July.
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2.2.
Experimental animals and management
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At a private sheep and goat farm in suburban area of Lahore there were 2483
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animals including 1219 sheep and 1264 goats of two breeds each of sheep (Dorper
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All Animals were kept under similar managemental/housing conditions of shades with
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grassy grounds and free access to water and fodder. Animals of each breed were kept
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in individual barrens separated by wall/fence. Animals were offered seasonal green
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fodder such as Medicago sativa (alfalfa/Lucerne) supplemented with formulated
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concentrate. All the flock was allowed grazing once a day for 4-6 hours.
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Animals were vaccinated against pleuropneumonia, peste des petits ruminants
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and enterotoxaemia and dewormed twice a year. Before this outbreak, 40 Kajli sheep
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and 25 Beetal goats were purchased from the local market, quarantined for a week,
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vaccinated with PESTDOLL-S® Freeze dried live attenuated PPR Virus strain
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Nigeria 75/1 Vaccine (M/S Dollvet Veterinary Vaccine, Medicine and Biological
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Materials Production Inc, Eyyübiye/ŞANLIURFA, Turkey) and then allowed to join
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the herd. Animals were routinely inspected for any disease and suspected animals
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were separated from rest of the animals while. They were handled by the same
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workers of the farm.
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2.3.
Morality
During hot and humid month, i.e., July (average temperature 40 °C with high
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humidity (81 %), animals started becoming sick and mortality started. Morbidity and
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mortality reached to 48.77 % and 21.50 %, respectively (Table 1).
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2.4.
Gross and histopathological study
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Postmortem was conducted immediately after the death of the sheep and goat.
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As the lesions in sheep (Dorper and Kajli) and goats (Boer and Beetal) were the same,
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therefore, a common picture of lesions was developed. Gross lesions were recorded
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and classified based on severity as mild, moderate and severe [40]. Morbid organs
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were put into 10 % neutral buffered formalin to prevent postmortem changes and for
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fixation. The tissues were completely immersed in formalin solution and kept for 10
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days at room temperature for proper fixation. Then tissues were subjected to washing,
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dehydration in ascending concentrations of alcohol, clearing in xylene, impregnation
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and embedding in paraffin [41]. Tissues were further processed by paraffin sectioning
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and 4-5 µm thick sections were stained with hematoxylin and eosin staining technique
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[42-44]. Slides were examined for histopathological changes by two pathologists, and
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if any difference was found, a third pathologist was consulted for opinion.
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2.5.
Detection of PPRV nucleic acid through RT-PCR
167 For PCR analysis, tissue samples of liver, lung, spleen, mesenteric and
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bronchial lymph nodes were collected in sterilized zip bags, kept in ice pack
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containers and transported to the laboratories for further processing. Pooled tissues of
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each organ were macerated using PBS as 20 % (w/v) suspension and centrifuged at
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800 g for 10 minutes. The supernatant was collected in sterile Falcon tubes,
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gentamycin (500 µg/mL) was added and stored at -20°C. Freeze-dried live PPR
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vaccine from Veterinary Research Institute was used as the positive controls. From
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the homogenates, RNA was extracted from the tissue suspension and vaccine virus
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using RNeasy Kit (Qiagen, Germany) as recommended by the manufacturer
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homogenate [45].
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PPRV nucleic acid was determined through RT-PCR using Qiagen One-Step
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RT-PCR kit and the NP3 (5´-TCTCGGAAATCGCCTCACAGACTG -3´) and NP4
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(5´-CCTCCTCCTGGTCCTCCAGAATCT -3´) primers for the amplification of a
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352-bp fragment of the PPRV nucleoprotein gene [46]. Briefly, RNA (5 µL) was
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amplified in a reaction mix (45 µL) containing the following: 5× Qiagen buffer (10
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µL), dNTP mix (2 µL), of Q-solution (10 µL), and One-Step RT-PCR mix (2 µL),
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each primer at a final concentration (0.6 µM) and water. First reverse transcription of
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RNA was done by incubating at 50°C for 30 minutes, then the PCR started with an
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initial denaturation and activation of Taq polymerase at 95°C for 15 minutes. This
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was followed 40 cycles of amplification corresponding to 30 s at 94°C/30 s at 60°C/1
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min at 72°C and a final extension of 10 min at 72°C. Ten microliters of the amplified
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product was analyzed by electrophoresis in 1.5 % agarose gel and results were
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recorded through gel documentation system.
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2.6.
Differential Diagnosis with other Infectious Diseases
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It is worth mentioning that last case of rinderpest (cattle plague) in Pakistan
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was diagnosed in September 2000. In November 2000, vaccination against rinderpest
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was officially prohibited in Pakistan [47]. According to FAO and so many other
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workers [48], now this globe is free from rinderpest, moreover, this was the disease of
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large ruminants, however, small ruminants are being affected by PPR. PPR is being
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covered in the present study. Other infections like Foot and Mouth disease and Bluetongue disease can
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easily be differentiated from PPR based on clinical signs and symptoms. Foot and
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Mouth disease occurs in sheep and goats, however, severity of FMD in sheep and
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goats is variable, usually mild. Mouth lesions are often transient and blisters like and
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most characteristics is lameness due to foot lesions, these lesions are absent in PPR
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[49]. Bluetongue disease also effects sheep and goats. Affected animals show
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swelling of lips, muzzle, and oral mucosa, cyanosis of the tongue together with edema
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of the head region - sufficient to differentiate Bluetongue from PPR. Coronitis is also
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not feature of PPR [49]. On these basis, other infectious diseases were ruled out in the
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present study.
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2.7.
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Data collected were subjected to the Chi-square test, using the Minitab statistical software package [47]. The significance level was P < 0.05.
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3.
Results
3.1.
Morbidity, mortality and case fatality
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Out of 2483 sheep and goats, 48.77 % animals become morbid and 21.50 %
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died (Table 1). Overall, case fatality was 44.10 %. Morbidity and mortality were
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significantly (P < 0.001) higher in indigenous breeds viz. Kajli sheep (75.37 % and
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32.80 %) and Beetal goat (81.10 % and 37.24 %) as compared to exotic breeds, i.e.,
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Dorper sheep (6.99 % and 1.48 %) and Australian Boer goats (5.01 % and 2.23 %).
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However, case fatality did not vary among sheep and goat breeds (Table 1).
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3.2.
Clinical signs
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Affected animals showed elevated body temperature (40–41 °C) which
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remained so for 3-5 days. These were anorexic with dull coat, depressed and had dry
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muzzle. Affected animals also showed nasal discharge, lachrymation and catarrhal
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advanced stages. Morbid animals showed rales and abdominal breathing with
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respiratory distress. Severe watery or blood-stained diarrhea along with dehydration,
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emaciation and dyspnea and finally death were common features in affected animals.
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3.3.
Gross lesions of Gastro-intestinal tract
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With the onset of fever, gums became hyperemic, and erosive lesions developed on
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the oral mucosa; dental pad and tongue with excessive salivation along with necrotic
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stomatitis and erosions of visible mucous membranes.
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Erosive lesions on the hard palate, pharynx and upper third of the esophagus
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were observed. Main lesions in the gastro-intestinal tract were i) small streaks of
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hemorrhages and occasional erosions in the first portion of the duodenum and the
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terminal ileum, ii) hemorrhagic or necrotic enteritis with extensive necrosis (Fig. 1a)
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and sometimes severe ulceration of Peyer's patches, iii) congestion around the ileo-
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cecal valve, at the ceco-colic junction and in the rectum and iv) zebra stripes of
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congestion in the posterior part of colon (Fig. 1b). Congestion and zebra stripping
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were the lesions most frequently seen around the ileo-cecal valve, at the ceco-colic
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junction, rectum and colon. Congestion and enlargement of mesenteric lymph nodes,
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spleen and liver were also observed.
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3.4.
Histopathological lesions of Gastro-intestinal tract
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Characteristic histopathological lesions of PPR were noted in the intestines,
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lymphoid organs, liver and lungs. The intestines showed stunting and blunting of villi
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(Fig. 2a) with necrosis of tips of the villi, as well as sloughing of the villi (Fig. 2b).
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Lamina propria showed infiltration of mononuclear cells and a few neutrophils (Fig.
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2a). Small intestines also exhibited diffuse edema of the submucosa along with
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proliferation of fibrocytes, increasing thickness of submucosa (Fig. 2a & 2b).
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Degeneration and necrosis of glandular epithelial cells, particularly in the upper
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duodenum, jejunum and ileum, was also seen.
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Peyer’s patches showed partial or complete destruction of lymphoid follicles
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characterized by lympholysis and karyorrhexis. Goblet cells hyperplasia and
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The spleen showed marked depletion of lymphocytes of parafollicular areas in the
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white pulp, together with reticuloendothelial cell hyperplasia (Fig. 3a). Sinusoids
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were dilated and filled with macrophages and plasma cells, with a few neutrophils
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(Fig. 3b). Mesenteric and retropharyngeal lymph nodes and tonsils, showed lesions
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similar to those of the spleen. Liver showed multifocal areas of coagulative necrosis, vacuolation of
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hepatocytes along with dilatation of sinusoidal spaces and congestion. Many
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hepatocyte nuclei in such foci were pyknotic. The periportal area, exhibited
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infiltration of inflammatory cells, mostly neutrophils, and few mononuclear cells
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along with engorged blood vessels (Fig. 4a).
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3.5.
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Lesions in the Kidneys
Kidneys showed severe degenerative changes including degeneration, severe
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necrosis (Fig. 4b-d), vacuolation and desquamation of epithelium of renal tubules
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(Fig. 4b-c). Glomerular hemorrhages and hyaline cast in the tubular lumen were also
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recorded (Fig. 4b-c).
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Gross lesions of respiratory tract
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Lesions of the respiratory tract were suggestive of bronchopneumonia,
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however, main lesions were i) small erosions and petechiae on the nasal mucosa and
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larynx, ii) severe congestion of trachea (Fig. 5a) along with froth (Fig. 5b) and severe
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congestion of apical lobe of lungs (Fig. 6a-c), and iii) deposition of fibrinous material
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in the lungs (Fig. 6d).
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Histopathological lesions of respiratory tract
The histopathological lesions in the lungs were more severe and characteristic
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of broncho interstitial pneumonia. There was desquamation of bronchiolar
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epithelium with the presence of epithelial cell debris in the lumen of bronchioles
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which was indicative of bronchopneumonia (Fig. 7a). The alveoli showed edema tour
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fluid mixed with fibrinous exudate, alveolar macrophages, moderate to high numbers
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of neutrophils (Fig. 7b-d) along with thickened interalveolar septa (Fig. 7b), indicative
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of interstitial pneumonia. Infiltration of mononuclear cells in alveoli, along with
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increased histiocyte proliferation was evident (Fig. 7). Fibrinous pneumonic lesions
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were accompanied by a serofibrinous exudate, with few macrophages and neutrophils
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in the alveolar lumina and presence of intranuclear eosinophilic inclusions (Fig. 8).
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3.8.
RT-PCR
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Gel electrophoresis for peste des petits ruminants one-step RT-PCR using NP3
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and NP4 primers showed a product of 352 bp size in spleen, lungs and mesenteric and
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brachial lymph node samples, while liver samples did not yield any results (Fig. 9).
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4.
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PPR studies usually orbit sero-epidemiology [8,10,12,36,37,51] and rare
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efforts have been made to report its pathology. Pathology of PPR has been reported in
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different goat and sheep breeds following experimental infection [18,38,52] but none
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in Dorper or Kajli sheep and Boer or Beetal goats. For the diagnosis of PPR, clinical
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and post-mortem findings may be sufficient [53], histopathological observations and
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molecular diagnosis or isolation of virus described in this article will further
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strengthen the diagnosis [52]. According to Durrani et al. [54], there could be three
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methods to diagnose and monitor the distribution and prevalence of PPR i.e., case
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recording of PPR outbreaks, detection of the virus and serological detection of PPR
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specific antibodies. In the present study, first two methods, in addition to gross and
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histopathological lesions recording were followed.
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Overall morbidity and mortality in the present study was 48.77 % and 21.50
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%, respectively (Table 1). Morbidity and mortality were significantly higher (P <
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0.001) in indigenous breed of sheep (Kajli) and goat (Beetal) as compared to imported
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breeds (Dorper sheep; Boer goat). Dorper sheep and Boer goats are Australian breeds.
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PPR rendered low morbidity and mortality in animals of these imported breeds,
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probably due to the fact that PPR is exotic to Australia [55,56] and these breeds could
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be resistant to PPR virus [57]. Moreover, Dorper sheep have a high degree of
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disease resistance [58], this could be the other reason that morbidity and mortality was
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significantly lower (P < 0.001) in Dorper sheep as compared to local sheep breed
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(Kajli). In Pakistan, PPR appeared in 1991 for the first time in the Punjab province
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[59,60]. Despite vaccination, PPR spread to all parts of the country [9,12-15,54,61-
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63]. The high susceptibility of local sheep and goat breeds to PPR virus [64] could be
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due to ineffective quarantine, continuous spread of PPR virus in non-vaccinated
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population, unrestricted transportation, absence of passive and active surveillance
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[8,16]. In the present study, morbidity, mortality and case fatality rates did not vary
337
significantly in sheep and goats (Table 1). Previous studies have reported significantly
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higher PPR infection rate in sheep as compared to goats [64]. Other studies reported
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higher prevalence of PPRV in goats than sheep [30,54,65-68]. The low prevalence of
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PPRV in sheep might be due to innate immune resistance [69]. The increased
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susceptibility of goats to PPRV than sheep can be due to less transmission of infection
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between these animals. Higher enzooticity of PPRV in goats than sheep could also be
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due to more adaptation and change in virulence of virus in goats which favors the
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spread of infection [30].
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Animals suffered with PPR showed high fever (40–41 °C) and serous nasal
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discharge in early stage of the disease, which became mucopurulent, dull coat,
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restless, depression, dry muzzle having lachrymation and catarrhal conjunctivitis in
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the present study. Moreover, morbid animals showed dyspnea, respiratory distress,
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severe watery or blood-stained diarrhea along with dehydration, emaciation and
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finally death. Various clinical signs such as anorexia, fever, depression, emaciation,
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stomatitis, occulo-nasal discharge, coughing, respiratory distress and death in
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naturally and experimentally infected goats have been reported earlier [70-72].
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The pathogenesis of PPRV is poorly understood, most of the information
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being based on comparison with related Morbilliviruses [73]. PPRV concentration is
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high in the exhaled air and body fluids i.e. saliva, oral and nasal discharges, urine,
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feces of the infected animals [74]. PPRV is primarily transmitted via the respiratory
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route among animals living in close proximity [75].
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Respiratory lesions in our study could be due to damage to respiratory mucosa
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(naso-pharyngeal/respiratory epithelium) [73,76]. It is reported that PPRV spreads to
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different other tissues of the body via regional lymphoid, then infects the lymphocytes
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and infection spreads throughout the body via both the lymphatic and vascular
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systems [71]. As the PPRV is both lympho- and epithelio-tropic [73], infection
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usually results in conjunctivitis, rhinotracheitis, ulcerative stomatitis, gastroenteritis
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and pneumonia which have been observed in the present study.
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At necropsy, main lesions were seen in the gastro-intestinal tract such as small
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and rectum along with severe congestion and enlargement of spleen and regional
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lymph nodes. These lesions might be due to extensive necrosis in lymphoid organs
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leading to inability of the animals to mount specific immune response to PPRV [76].
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In the present study, fragile liver, enlarged lymph nodes, severe enteritis along with
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streaks of hemorrhages ‘zebra stripes’, and enlargement and petechial hemorrhages in
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the spleen were observed which have also been reported previously [18,19,29].
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Different pulmonary lesions observed in the present study including small
375
erosions on the nasal mucosa and larynx, congestion, petechiae, hemorrhages and
376
frothy exudates in the trachea, red hepatization, raised patches of emphysema in the
377
lungs, severe congestion of apical lobe of lungs and presence of fibrinous exudate in
378
the lungs have been reported in PPR infected sheep, goats [18] and camels [77].
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At histopathological level, main lesions in the intestine such as stunting and
380
blunting of villi along with necrosis and sloughing of the villi could be due to
381
cytopathogenicity of PPRV, leading to necrosis/apoptosis. Squamous epithelial
382
syncytia are also observed in digestive tract epithelium following PPR infection
383
[73,74]. In the present study, small intestine also exhibited diffuse edema of the
384
submucosa along with proliferation of fibrocytes, leading to thickened submucosa
385
which has not been reported in the published literature as far as our knowledge is
386
concerned.
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In the present study, Peyer’s patches, spleen and lymph nodes in PPR affected
388
animals showed lympholysis, necrotic changes and depletion of lymphoid cells along
389
with hyperplasia of reticuloendothelial cells. Like Morbillivirus, PPRV has great
390
affinity to lymphoid cells [29,72], causing damage to the lymphoid organs [72,74,78].
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Histologically, liver of sheep died of PPR in the present study showed
392
multifocal necrotic areas in the present study which have been reported in the
393
published literature [49,79]. Moreover, coagulative necrosis, multifocal vacuolation of
394
hepatocytes, perivascular mononuclear cellular infiltration, especially in portal areas,
395
and macrophage aggregations within parenchyma have been reported [72].
396
Histological analysis of liver tissues in the present study showed engorged blood
397
vessels, dilation of sinusoidal spaces, hemorrhages and infiltration of neutrophils and
398
mononuclear cells, particularly in periportal areas [18].
399
Kidneys also showed degenerative changes in infected animals in the present
400
study. Severe necrosis, vacuolation, degeneration and desquamation of tubular Page 12 of 23
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epithelium, glomerular hemorrhages and hyaline tubular cast in the tubular lumen
402
have been reported in kidneys of infected goats [18]. Kidneys revealed congestion in
403
glomeruli and cortical blood vessels [70]. As the PPRV is primarily transmitted via the respiratory route [75], this tract is
405
severely affected. In our study, lesions like broncho interstitial pneumonia,
406
bronchopneumonia, extensive alveolar edema mixed with fibrinous exudate,
407
interstitial pneumonia characterized by thickened interalveolar septa and fibrinous
408
pneumonia were recorded in affected animals. Presence of intranuclear eosinophilic
409
inclusions in alveolar macrophages was another important feature noted in affected
410
animals, as reported previously [29,49,72]. Available published literature shows that
411
upon exposure to virulent PPRV, susceptible animals usually develop acute
412
pulmonary congestion and edema, leading to bronchopneumonia and even diffused
413
pneumonia, then succumb to death [19,73]. In the same sequence the events in the
414
respiratory tract of died animals took place in the present study.
415 416
Conflict of interest
417
Authors have stated no competing interests.
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T.U. Obi, M.O. Ojo, O.A. Durojaiye, O.B. Kasali, S. Akparie, D.B. Opasina,
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Peste des petits ruminants in goats in Nigeria: clinical, microbiological and
664
pathological features, Zentralbl Veterinarmed B. 30 (1983) 751–761.
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Table 1: Morbidity, mortality and case fatality in sheep and goats due to peste des
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petits ruminants Breeds
Total
*Morbidity
Animals
N
**Mortality
%
N
%
***Case Fatality (%)
Sheep breeds 472
33
6.99
7
Kajli
747
563
75.37
245
Dorper vs Kajli sheep
P Value = 0.001
P Value = 0.001
P Value = 0.083
5.01
Beetal
725
588
81.10
12
2.23
44.44
270
37.24
45.92
χ2 Value = 148.116
χ2 Value = 0.008
P Value = 0.001
P Value = 0.001
P Value = 0.927
Sheep
1219
596
Goats
1264
615
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χ2 Value = 287.594
48.89
252
20.67
42.28
48.65
282
22.31
45.85
χ2 Value = 0.005
χ2 Value = 0.637
χ2 Value = 0.608
P Value = 0.945
P Value = 0.425
P Value = 0.436
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2483
43.52
χ2 Value = 3.000
539
Total/Overall
32.80
χ2 Value = 122.853
Boer
Sheep vs Goats
21.21
χ2 Value = 219.485
Goat breeds
Boer vs Beetal goats
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48.77
534
21.50
44.10
Data analysis by Chi-square test and df in each case was 1.
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Number of animals became sick
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Total number of animals at risk
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Number of animals died
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***Case Fatality (%) = 672
Total number of animals at risk
X 100
Number of animals died Number of animals became sick
X 100
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Captions of Figures
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Fig. 1:
Photographs of intestines of a) a sheep and b) a goat died of peste des petits
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ruminants showing streaks of hemorrhages (zebra stripping) and blood clots
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(arrows). Fig. 2:
Photomicrographs of intestines of a goat (a and b) died of peste des petits
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ruminants, showing stunting and blunting of villi (arrows) with necrosis of
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tips of the villi, as well as sloughing of the villi (arrow head). There is
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infiltration of the lamina propria with mononuclear cells and a few
682
neutrophil leukocytes (asterisk) along with diffuse edema of the submucosa
683
and proliferation of fibrocytes, forming the submucosa much thickened
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(double-head arrow). H and E Stain. X 100. Fig. 3:
Photomicrographs of spleen of a sheep (a and b) died of peste des petits
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ruminants, showing marked depletion of lymphocytes in the white pulp,
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together with reticuloendothelial cell hyperplasia (arrow heads) and
688
congestion (arrow). Dilated sinusoidal spaces are filled with macrophages
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and plasma cells, with a few neutrophils. H and E Stain. X 100. Fig. 4:
Photomicrograph of liver (a) of a goat died of peste des petits ruminants,
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showing congestion (arrow head), cellular infiltration (arrows) and many
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hepatocytes have under gone degeneration. b-d) kidneys of sheep and goat
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died of peste des petits ruminants showing renal tubular necrosis (arrows),
694
detachment of tubular epithelium from the basement membrane and pooled
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in the lumen (arrow heads) and filling of renal tubules with proteinaceous
696
material and casts (asterisks). H and E Stain. a) X 40; b & d) X 200 and c)
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X400.
Fig. 5:
Photograph of trachea of a sheep died of peste des petits ruminants, showing a) severe congestion and b) severe congestion with froth.
Fig. 6:
Photographs of lungs of a goat died of peste des petits ruminants showing
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severe congestion of apical lobe of the lungs (a-c: arrows) and deposition of
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fibrinous materials (d: arrow heads).
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Fig. 7:
Photomicrograph of lungs of a sheep died of peste des petits ruminants
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showing a) alveoli filled with serofibrinous edema fluid mixed with
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fibrinous exudate (asterisks), numerous alveolar macrophages and moderate
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thickened interalveolar septa (arrows) and extensive infiltration of
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mononuclear cells in alveoli (arrow heads); c & d) characteristic interstitial
709
pneumonia with extensive infiltration of erythrocytes, mononuclear cells and
710
increased histiocyte proliferation. H and E Stain. a-c) X 100 and d) X 400.
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Fig. 8:
Photomicrographs of lungs of a goat died of peste des petits ruminants showing lesions of bronchopneumonia. a-b) alveoli filled with serofibrinous
713
edema fluid mixed with fibrinous exudate (asterisks), congestion (arrows),
714
numerous alveolar macrophages and moderate numbers of neutrophil
715
leukocytes in alveoli; and intranuclear eosinophilic inclusions in alveolar
716
macrophages (arrows). H and E Stain. X400. Fig. 9:
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Photograph of Gel electrophoresis for peste des petits ruminants one-step RT-PCR using NP3 and NP4 primers (product size = 352 bp). Lane 1 =
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Ladder (bp); Lane 2 = Spleen sample; Lane 3 = Negative control; Lane 4 =
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Lungs sample; Lane 5 = Mesenteric lymph node sample; Lane 6 = Brachial
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lymph node sample; Lane 7 = Liver sample and Lane 8 = Positive control.
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Highlights of the manuscript Pathophysiology of Peste Des Petits Ruminants in Sheep (Dorper & Kajli) and Goats (Boer & Beetal) •
Peste des petits ruminants was studied in local (Kajli sheep; Beetal goats) as well as
•
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imported small ruminant breeds (Dorper sheep; Australian Boer goat).
Morbidity and mortality rates were significantly higher in indigenous Kajli sheep and Beetal goats as compared to Dorper sheep and Australian Boer goat.
•
On postmortem examination, lungs were congested and pneumonic, with nodular and
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cystic appearance and intestines were hemorrhagic with zebra stripping.
Histopathological lesions of respiratory tract were severe and characteristic of broncho
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One-Step RT-PCR using NP3 and NP4 primers confirmed a PPR virus of 352 bp size in
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S0882-4010(18)30008-1
DOI:
10.1016/j.micpath.2018.02.009
Reference:
YMPAT 2775
To appear in:
Microbial Pathogenesis
Received Date: 2 January 2018 Revised Date:
4 February 2018
Accepted Date: 6 February 2018
Please cite this article as: Khan A, Saleemi MK, Ali F, Abubakar M, Hussain R, Abbas RZ, Khan IA, Pathophysiology of peste des petits ruminants in sheep (Dorper & Kajli) and goats (Boer & Beetal), Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.02.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Pathophysiology of Peste Des Petits Ruminants in Sheep (Dorper & Kajli) and
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Goats (Boer & Beetal)
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Ahrar Khan1*, M. Kashif Saleemi1, Farah Ali2, Muhammad Abubakar3, Riaz
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Hussain2, Rao Zahid Abbas1 and Imtiaz Ahmad Khan4
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Pakistan.
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Faculty of Veterinary Science, University of Agriculture, Faisalabad-38000,
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Bahawalpur- 63100, Pakistan.
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University College of Veterinary and Animal Sciences, The Islamia University of
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National Veterinary Laboratory, Islamabad, Pakistan
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Department of Pathobiology, PMAS-Arid Agriculture University, Rawalpindi-
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46000, Pakistan
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17 Corresponding Author:
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Ahrar Khan (
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Department of Pathology,
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Faculty of Veterinary Science,
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University of Agriculture,
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Faisalabad-38000, Pakistan;
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[email protected]; [email protected]
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Phone: +92 333 651 7844
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https://orcid.org/0000-0001-5492-426)
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Page 1 of 23
ACCEPTED MANUSCRIPT Abstract
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Peste des petits ruminants (PPR), an economically important viral transboundary
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disease of small ruminants is not only prevalent in Pakistan but also in other countries
31
where people rely on agriculture and animal products. The present study was aimed at
32
describing the pathology and antigen localization in natural PPR infections in local
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(Kajli sheep; Beetal goats) as well as imported small ruminant breeds (Dorper sheep;
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Australian Boer goat). Morbidity and mortality rates were significantly (P < 0.001)
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higher in indigenous Kajli sheep (75.37 and 32.80 %) and Beetal goats (81.10 and
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37.24 %) as compared to Dorper sheep (6.99 and 1.48 %) and Australian Boer goat
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(5.01 and 2.23 %). Affected animals exhibited high fever, severe diarrhea, abdominal
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pain, respiratory distress and nodular lesions on lips and nostrils. Thick mucous
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discharge was oozing out from nostrils. On postmortem examination, lungs were
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congested and pneumonic, with nodular and cystic appearance and intestines were
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hemorrhagic with zebra stripping. Characteristic histopathological lesions of PPR
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were noted in intestines, lymphoid organs and lungs. In GI tract, stunting and blunting
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of villi, necrotic enteritis, and infiltration of mononuclear cells in duodenum, jejunum
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and ileum. Small intestines exhibited diffuse edema of the submucosa along with
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proliferation of fibrocytes, leading to thickened submucosa which has not been
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reported previously. Lymphoid organs showed partial to complete destruction of
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lymphoid follicles. Lesions of the respiratory tract included depictive of
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bronchopneumonia, severe congestion of trachea and apical lobe of lungs with
49
deposition of fibrinous materials. Histopathological lesions of respiratory tract were
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severe and characteristic of broncho interstitial pneumonia, bronchopneumonia,
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interstitial pneumonia and fibrinous pneumonia. The alveoli were filled with
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edematous fluid mixed with fibrinous exudate, numerous alveolar macrophages,
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mononuclear cells along with thickened interalveolar septa and presence of
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intranuclear eosinophilic inclusions. One-Step RT-PCR using NP3 and NP4 primers
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confirmed a PPR virus of 352 bp size in spleen, lungs and mesenteric and brachial
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lymph node samples. It was concluded that morbidity and mortality due to PPR were
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significantly higher in indigenous breeds of sheep and goat as compared to imported
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sheep and goat breeds. PPR has rendered various lesions in GI and respiratory tract
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which are characteristic in nature for the diagnosis of the disease under field
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condition.
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Keywords: Peste des petits ruminants, sheep, goat, morbidity, mortality, gross and
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histopathological lesions of GI and respiratory tract
64 65
1.
Introduction
66 The economy of Pakistan is agro-livestock based and role of livestock in agriculture
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sector is pivotal in rural socio-economic development [1-3]. Nearly eight million
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families in the country are involved in livestock raising. This sector is a source of
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income, and often the only source of income, for the rural and most marginal people,
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thus playing a key role in poverty alleviation [4,5]. Livestock sector is contributing
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approximately 58.6 % to the agriculture value added and 11.6 % to the overall GDP in
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Pakistan. Sheep and goats, being dual purpose animals with estimated population of
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70.3 and 29.8 million heads, respectively, are used for meat and to some extent for
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milk production [6].
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Sheep and goat farming in Pakistan is progressing rapidly and is contributing
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significantly to the national economy. However, its growth is being hampered by
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various diseases [7], including respiratory ailments like peste des petits ruminants
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[8,9]. Peste des petits ruminants (PPR) is reportable to the World Organization for
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Animal Health (OIE) and is an economically important disease in parts of Africa and
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some Asian countries. It is among transboundary diseases [10,11] and is not only
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present in Pakistan [12-15] but also in surrounding countries, including Afghanistan
83
[16,17], India [18-21], Iran [22] and China [23,24]. This disease has also been
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reported from east African and Middle East countries and from most third world
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countries where people rely on agriculture and animal products [25,26].
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PPR is primarily a disease of sheep and goat but it has also been reported in
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Ethiopian camels, Indian buffalos [27], Saudian gazelles and deers [28] etc. Peste des
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petits ruminants, also known as goat plague and ovine rinderpest, affects sheep, goats
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and related species of small ruminants [29] and is highly contagious viral disease
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[30,31]. Morbillivirus, the causative agent, is closely related to rinderpest, measles
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and canine distemper virus [17,32]. The principal pathological findings of PPR are
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seen in the digestive and respiratory systems [33,34]. Morbidity and mortality could
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be 5-90 and 50-80 %, respectively [35,36].
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Vaccination is a strategy to control PPR and the vaccine is required at mass
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scale, however, losses can be curtailed through preventive measures for which Page 3 of 23
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usually revolve around sero-epidemiology [12,36,37] and rare efforts have been made
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to report its pathology. Most of the available information on the pathology of PPR has
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been based on results obtained following experimental infections [38] but none in
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Dorper or Kajli sheep and Boer or Beetal goats under field conditions. Data regarding
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pathological parameters from field cases are very important as these can better
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characterize the pathogenicity of the circulating virus. However, such data of PPR in
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Dorper/Kajli sheep and Boer/Beetal goats are lacking in the published literature. The
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severity of the disease varies with species, immunity and breed of the host [29]. The
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present study was aimed at describing the pathology and antigen localization in
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natural PPR infections in exotic sheep and goat breeds (Boer goat and Dorpar sheep)
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kept in close contact with local sheep (Kajli) and goat (Beetal) breeds.
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2.
Materials and methods
2.1.
Study Area and Environment
Lahore is the capital city of the Punjab province of Pakistan. It is the second most populous city in Pakistan and the 32nd most populous city in the world [39]. The
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city is located in the north-eastern end of the province, bordering with the Indian state
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of Punjab. Lahore has a semi-arid climate. Lying between 31°15′ - 31°45′ N and
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74°01′ - 74°39′ E, Lahore is bounded on the north and west by the Sheikhupura
118
District, on the east by Wagah, and on the south by Kasur District. Study area was in
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suburban of Lahore towards Kasur District. Here, the hottest month of the year is
120
June, with an average temperature of 40 °C (104.0 °F) and the coldest month is
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January at 14°C (56°F) with dense fog [1]. The monsoon season starts in late June,
122
and the hot and humid month is July with heavy rainfalls, high humidity (81 %) and
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evening thunderstorms with the possibility of cloudbursts. This study was conducted
124
in suburban of Lahore towards Kasur District during the mid of July.
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2.2.
Experimental animals and management
127 128
At a private sheep and goat farm in suburban area of Lahore there were 2483
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animals including 1219 sheep and 1264 goats of two breeds each of sheep (Dorper
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All Animals were kept under similar managemental/housing conditions of shades with
133
grassy grounds and free access to water and fodder. Animals of each breed were kept
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in individual barrens separated by wall/fence. Animals were offered seasonal green
135
fodder such as Medicago sativa (alfalfa/Lucerne) supplemented with formulated
136
concentrate. All the flock was allowed grazing once a day for 4-6 hours.
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Animals were vaccinated against pleuropneumonia, peste des petits ruminants
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and enterotoxaemia and dewormed twice a year. Before this outbreak, 40 Kajli sheep
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and 25 Beetal goats were purchased from the local market, quarantined for a week,
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vaccinated with PESTDOLL-S® Freeze dried live attenuated PPR Virus strain
141
Nigeria 75/1 Vaccine (M/S Dollvet Veterinary Vaccine, Medicine and Biological
142
Materials Production Inc, Eyyübiye/ŞANLIURFA, Turkey) and then allowed to join
143
the herd. Animals were routinely inspected for any disease and suspected animals
144
were separated from rest of the animals while. They were handled by the same
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workers of the farm.
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2.3.
Morality
During hot and humid month, i.e., July (average temperature 40 °C with high
149
humidity (81 %), animals started becoming sick and mortality started. Morbidity and
150
mortality reached to 48.77 % and 21.50 %, respectively (Table 1).
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2.4.
Gross and histopathological study
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Postmortem was conducted immediately after the death of the sheep and goat.
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As the lesions in sheep (Dorper and Kajli) and goats (Boer and Beetal) were the same,
156
therefore, a common picture of lesions was developed. Gross lesions were recorded
157
and classified based on severity as mild, moderate and severe [40]. Morbid organs
158
were put into 10 % neutral buffered formalin to prevent postmortem changes and for
159
fixation. The tissues were completely immersed in formalin solution and kept for 10
160
days at room temperature for proper fixation. Then tissues were subjected to washing,
161
dehydration in ascending concentrations of alcohol, clearing in xylene, impregnation
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and embedding in paraffin [41]. Tissues were further processed by paraffin sectioning
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and 4-5 µm thick sections were stained with hematoxylin and eosin staining technique
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[42-44]. Slides were examined for histopathological changes by two pathologists, and
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if any difference was found, a third pathologist was consulted for opinion.
166
2.5.
Detection of PPRV nucleic acid through RT-PCR
167 For PCR analysis, tissue samples of liver, lung, spleen, mesenteric and
169
bronchial lymph nodes were collected in sterilized zip bags, kept in ice pack
170
containers and transported to the laboratories for further processing. Pooled tissues of
171
each organ were macerated using PBS as 20 % (w/v) suspension and centrifuged at
172
800 g for 10 minutes. The supernatant was collected in sterile Falcon tubes,
173
gentamycin (500 µg/mL) was added and stored at -20°C. Freeze-dried live PPR
174
vaccine from Veterinary Research Institute was used as the positive controls. From
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the homogenates, RNA was extracted from the tissue suspension and vaccine virus
176
using RNeasy Kit (Qiagen, Germany) as recommended by the manufacturer
177
homogenate [45].
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PPRV nucleic acid was determined through RT-PCR using Qiagen One-Step
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RT-PCR kit and the NP3 (5´-TCTCGGAAATCGCCTCACAGACTG -3´) and NP4
180
(5´-CCTCCTCCTGGTCCTCCAGAATCT -3´) primers for the amplification of a
181
352-bp fragment of the PPRV nucleoprotein gene [46]. Briefly, RNA (5 µL) was
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amplified in a reaction mix (45 µL) containing the following: 5× Qiagen buffer (10
183
µL), dNTP mix (2 µL), of Q-solution (10 µL), and One-Step RT-PCR mix (2 µL),
184
each primer at a final concentration (0.6 µM) and water. First reverse transcription of
185
RNA was done by incubating at 50°C for 30 minutes, then the PCR started with an
186
initial denaturation and activation of Taq polymerase at 95°C for 15 minutes. This
187
was followed 40 cycles of amplification corresponding to 30 s at 94°C/30 s at 60°C/1
188
min at 72°C and a final extension of 10 min at 72°C. Ten microliters of the amplified
189
product was analyzed by electrophoresis in 1.5 % agarose gel and results were
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recorded through gel documentation system.
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2.6.
Differential Diagnosis with other Infectious Diseases
193
It is worth mentioning that last case of rinderpest (cattle plague) in Pakistan
194
was diagnosed in September 2000. In November 2000, vaccination against rinderpest
195
was officially prohibited in Pakistan [47]. According to FAO and so many other
196
workers [48], now this globe is free from rinderpest, moreover, this was the disease of
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large ruminants, however, small ruminants are being affected by PPR. PPR is being
198
covered in the present study. Other infections like Foot and Mouth disease and Bluetongue disease can
200
easily be differentiated from PPR based on clinical signs and symptoms. Foot and
201
Mouth disease occurs in sheep and goats, however, severity of FMD in sheep and
202
goats is variable, usually mild. Mouth lesions are often transient and blisters like and
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most characteristics is lameness due to foot lesions, these lesions are absent in PPR
204
[49]. Bluetongue disease also effects sheep and goats. Affected animals show
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swelling of lips, muzzle, and oral mucosa, cyanosis of the tongue together with edema
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of the head region - sufficient to differentiate Bluetongue from PPR. Coronitis is also
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not feature of PPR [49]. On these basis, other infectious diseases were ruled out in the
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present study.
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2.7.
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Data collected were subjected to the Chi-square test, using the Minitab statistical software package [47]. The significance level was P < 0.05.
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3.
Results
3.1.
Morbidity, mortality and case fatality
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died (Table 1). Overall, case fatality was 44.10 %. Morbidity and mortality were
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significantly (P < 0.001) higher in indigenous breeds viz. Kajli sheep (75.37 % and
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32.80 %) and Beetal goat (81.10 % and 37.24 %) as compared to exotic breeds, i.e.,
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Dorper sheep (6.99 % and 1.48 %) and Australian Boer goats (5.01 % and 2.23 %).
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However, case fatality did not vary among sheep and goat breeds (Table 1).
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Clinical signs
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Affected animals showed elevated body temperature (40–41 °C) which
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remained so for 3-5 days. These were anorexic with dull coat, depressed and had dry
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advanced stages. Morbid animals showed rales and abdominal breathing with
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respiratory distress. Severe watery or blood-stained diarrhea along with dehydration,
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emaciation and dyspnea and finally death were common features in affected animals.
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3.3.
Gross lesions of Gastro-intestinal tract
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With the onset of fever, gums became hyperemic, and erosive lesions developed on
239
the oral mucosa; dental pad and tongue with excessive salivation along with necrotic
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stomatitis and erosions of visible mucous membranes.
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Erosive lesions on the hard palate, pharynx and upper third of the esophagus
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were observed. Main lesions in the gastro-intestinal tract were i) small streaks of
243
hemorrhages and occasional erosions in the first portion of the duodenum and the
244
terminal ileum, ii) hemorrhagic or necrotic enteritis with extensive necrosis (Fig. 1a)
245
and sometimes severe ulceration of Peyer's patches, iii) congestion around the ileo-
246
cecal valve, at the ceco-colic junction and in the rectum and iv) zebra stripes of
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congestion in the posterior part of colon (Fig. 1b). Congestion and zebra stripping
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were the lesions most frequently seen around the ileo-cecal valve, at the ceco-colic
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junction, rectum and colon. Congestion and enlargement of mesenteric lymph nodes,
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spleen and liver were also observed.
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3.4.
Histopathological lesions of Gastro-intestinal tract
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Characteristic histopathological lesions of PPR were noted in the intestines,
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lymphoid organs, liver and lungs. The intestines showed stunting and blunting of villi
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(Fig. 2a) with necrosis of tips of the villi, as well as sloughing of the villi (Fig. 2b).
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Lamina propria showed infiltration of mononuclear cells and a few neutrophils (Fig.
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2a). Small intestines also exhibited diffuse edema of the submucosa along with
259
proliferation of fibrocytes, increasing thickness of submucosa (Fig. 2a & 2b).
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Degeneration and necrosis of glandular epithelial cells, particularly in the upper
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duodenum, jejunum and ileum, was also seen.
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The spleen showed marked depletion of lymphocytes of parafollicular areas in the
266
white pulp, together with reticuloendothelial cell hyperplasia (Fig. 3a). Sinusoids
267
were dilated and filled with macrophages and plasma cells, with a few neutrophils
268
(Fig. 3b). Mesenteric and retropharyngeal lymph nodes and tonsils, showed lesions
269
similar to those of the spleen. Liver showed multifocal areas of coagulative necrosis, vacuolation of
271
hepatocytes along with dilatation of sinusoidal spaces and congestion. Many
272
hepatocyte nuclei in such foci were pyknotic. The periportal area, exhibited
273
infiltration of inflammatory cells, mostly neutrophils, and few mononuclear cells
274
along with engorged blood vessels (Fig. 4a).
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3.5.
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Lesions in the Kidneys
Kidneys showed severe degenerative changes including degeneration, severe
278
necrosis (Fig. 4b-d), vacuolation and desquamation of epithelium of renal tubules
279
(Fig. 4b-c). Glomerular hemorrhages and hyaline cast in the tubular lumen were also
280
recorded (Fig. 4b-c).
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281 3.6.
Gross lesions of respiratory tract
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Lesions of the respiratory tract were suggestive of bronchopneumonia,
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however, main lesions were i) small erosions and petechiae on the nasal mucosa and
285
larynx, ii) severe congestion of trachea (Fig. 5a) along with froth (Fig. 5b) and severe
286
congestion of apical lobe of lungs (Fig. 6a-c), and iii) deposition of fibrinous material
287
in the lungs (Fig. 6d).
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Histopathological lesions of respiratory tract
The histopathological lesions in the lungs were more severe and characteristic
292
of broncho interstitial pneumonia. There was desquamation of bronchiolar
293
epithelium with the presence of epithelial cell debris in the lumen of bronchioles
294
which was indicative of bronchopneumonia (Fig. 7a). The alveoli showed edema tour
295
fluid mixed with fibrinous exudate, alveolar macrophages, moderate to high numbers
296
of neutrophils (Fig. 7b-d) along with thickened interalveolar septa (Fig. 7b), indicative
297
of interstitial pneumonia. Infiltration of mononuclear cells in alveoli, along with
298
hemorrhages, caused by the infiltration of erythrocytes, mononuclear cells and Page 9 of 23
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increased histiocyte proliferation was evident (Fig. 7). Fibrinous pneumonic lesions
300
were accompanied by a serofibrinous exudate, with few macrophages and neutrophils
301
in the alveolar lumina and presence of intranuclear eosinophilic inclusions (Fig. 8).
302 303
3.8.
RT-PCR
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Gel electrophoresis for peste des petits ruminants one-step RT-PCR using NP3
306
and NP4 primers showed a product of 352 bp size in spleen, lungs and mesenteric and
307
brachial lymph node samples, while liver samples did not yield any results (Fig. 9).
309
4.
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308 Discussion
PPR studies usually orbit sero-epidemiology [8,10,12,36,37,51] and rare
311
efforts have been made to report its pathology. Pathology of PPR has been reported in
312
different goat and sheep breeds following experimental infection [18,38,52] but none
313
in Dorper or Kajli sheep and Boer or Beetal goats. For the diagnosis of PPR, clinical
314
and post-mortem findings may be sufficient [53], histopathological observations and
315
molecular diagnosis or isolation of virus described in this article will further
316
strengthen the diagnosis [52]. According to Durrani et al. [54], there could be three
317
methods to diagnose and monitor the distribution and prevalence of PPR i.e., case
318
recording of PPR outbreaks, detection of the virus and serological detection of PPR
319
specific antibodies. In the present study, first two methods, in addition to gross and
320
histopathological lesions recording were followed.
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Overall morbidity and mortality in the present study was 48.77 % and 21.50
322
%, respectively (Table 1). Morbidity and mortality were significantly higher (P <
323
0.001) in indigenous breed of sheep (Kajli) and goat (Beetal) as compared to imported
324
breeds (Dorper sheep; Boer goat). Dorper sheep and Boer goats are Australian breeds.
325
PPR rendered low morbidity and mortality in animals of these imported breeds,
326
probably due to the fact that PPR is exotic to Australia [55,56] and these breeds could
327
be resistant to PPR virus [57]. Moreover, Dorper sheep have a high degree of
328
disease resistance [58], this could be the other reason that morbidity and mortality was
329
significantly lower (P < 0.001) in Dorper sheep as compared to local sheep breed
330
(Kajli). In Pakistan, PPR appeared in 1991 for the first time in the Punjab province
331
[59,60]. Despite vaccination, PPR spread to all parts of the country [9,12-15,54,61-
332
63]. The high susceptibility of local sheep and goat breeds to PPR virus [64] could be
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due to ineffective quarantine, continuous spread of PPR virus in non-vaccinated
334
population, unrestricted transportation, absence of passive and active surveillance
335
[8,16]. In the present study, morbidity, mortality and case fatality rates did not vary
337
significantly in sheep and goats (Table 1). Previous studies have reported significantly
338
higher PPR infection rate in sheep as compared to goats [64]. Other studies reported
339
higher prevalence of PPRV in goats than sheep [30,54,65-68]. The low prevalence of
340
PPRV in sheep might be due to innate immune resistance [69]. The increased
341
susceptibility of goats to PPRV than sheep can be due to less transmission of infection
342
between these animals. Higher enzooticity of PPRV in goats than sheep could also be
343
due to more adaptation and change in virulence of virus in goats which favors the
344
spread of infection [30].
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Animals suffered with PPR showed high fever (40–41 °C) and serous nasal
346
discharge in early stage of the disease, which became mucopurulent, dull coat,
347
restless, depression, dry muzzle having lachrymation and catarrhal conjunctivitis in
348
the present study. Moreover, morbid animals showed dyspnea, respiratory distress,
349
severe watery or blood-stained diarrhea along with dehydration, emaciation and
350
finally death. Various clinical signs such as anorexia, fever, depression, emaciation,
351
stomatitis, occulo-nasal discharge, coughing, respiratory distress and death in
352
naturally and experimentally infected goats have been reported earlier [70-72].
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The pathogenesis of PPRV is poorly understood, most of the information
354
being based on comparison with related Morbilliviruses [73]. PPRV concentration is
355
high in the exhaled air and body fluids i.e. saliva, oral and nasal discharges, urine,
356
feces of the infected animals [74]. PPRV is primarily transmitted via the respiratory
357
route among animals living in close proximity [75].
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Respiratory lesions in our study could be due to damage to respiratory mucosa
359
(naso-pharyngeal/respiratory epithelium) [73,76]. It is reported that PPRV spreads to
360
different other tissues of the body via regional lymphoid, then infects the lymphocytes
361
and infection spreads throughout the body via both the lymphatic and vascular
362
systems [71]. As the PPRV is both lympho- and epithelio-tropic [73], infection
363
usually results in conjunctivitis, rhinotracheitis, ulcerative stomatitis, gastroenteritis
364
and pneumonia which have been observed in the present study.
365
At necropsy, main lesions were seen in the gastro-intestinal tract such as small
366
streaks of hemorrhages in duodenum, terminal ileum, hemorrhagic/necrotic enteritis Page 11 of 23
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and rectum along with severe congestion and enlargement of spleen and regional
369
lymph nodes. These lesions might be due to extensive necrosis in lymphoid organs
370
leading to inability of the animals to mount specific immune response to PPRV [76].
371
In the present study, fragile liver, enlarged lymph nodes, severe enteritis along with
372
streaks of hemorrhages ‘zebra stripes’, and enlargement and petechial hemorrhages in
373
the spleen were observed which have also been reported previously [18,19,29].
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Different pulmonary lesions observed in the present study including small
375
erosions on the nasal mucosa and larynx, congestion, petechiae, hemorrhages and
376
frothy exudates in the trachea, red hepatization, raised patches of emphysema in the
377
lungs, severe congestion of apical lobe of lungs and presence of fibrinous exudate in
378
the lungs have been reported in PPR infected sheep, goats [18] and camels [77].
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At histopathological level, main lesions in the intestine such as stunting and
380
blunting of villi along with necrosis and sloughing of the villi could be due to
381
cytopathogenicity of PPRV, leading to necrosis/apoptosis. Squamous epithelial
382
syncytia are also observed in digestive tract epithelium following PPR infection
383
[73,74]. In the present study, small intestine also exhibited diffuse edema of the
384
submucosa along with proliferation of fibrocytes, leading to thickened submucosa
385
which has not been reported in the published literature as far as our knowledge is
386
concerned.
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In the present study, Peyer’s patches, spleen and lymph nodes in PPR affected
388
animals showed lympholysis, necrotic changes and depletion of lymphoid cells along
389
with hyperplasia of reticuloendothelial cells. Like Morbillivirus, PPRV has great
390
affinity to lymphoid cells [29,72], causing damage to the lymphoid organs [72,74,78].
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Histologically, liver of sheep died of PPR in the present study showed
392
multifocal necrotic areas in the present study which have been reported in the
393
published literature [49,79]. Moreover, coagulative necrosis, multifocal vacuolation of
394
hepatocytes, perivascular mononuclear cellular infiltration, especially in portal areas,
395
and macrophage aggregations within parenchyma have been reported [72].
396
Histological analysis of liver tissues in the present study showed engorged blood
397
vessels, dilation of sinusoidal spaces, hemorrhages and infiltration of neutrophils and
398
mononuclear cells, particularly in periportal areas [18].
399
Kidneys also showed degenerative changes in infected animals in the present
400
study. Severe necrosis, vacuolation, degeneration and desquamation of tubular Page 12 of 23
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epithelium, glomerular hemorrhages and hyaline tubular cast in the tubular lumen
402
have been reported in kidneys of infected goats [18]. Kidneys revealed congestion in
403
glomeruli and cortical blood vessels [70]. As the PPRV is primarily transmitted via the respiratory route [75], this tract is
405
severely affected. In our study, lesions like broncho interstitial pneumonia,
406
bronchopneumonia, extensive alveolar edema mixed with fibrinous exudate,
407
interstitial pneumonia characterized by thickened interalveolar septa and fibrinous
408
pneumonia were recorded in affected animals. Presence of intranuclear eosinophilic
409
inclusions in alveolar macrophages was another important feature noted in affected
410
animals, as reported previously [29,49,72]. Available published literature shows that
411
upon exposure to virulent PPRV, susceptible animals usually develop acute
412
pulmonary congestion and edema, leading to bronchopneumonia and even diffused
413
pneumonia, then succumb to death [19,73]. In the same sequence the events in the
414
respiratory tract of died animals took place in the present study.
415 416
Conflict of interest
417
Authors have stated no competing interests.
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pathological features, Zentralbl Veterinarmed B. 30 (1983) 751–761.
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Table 1: Morbidity, mortality and case fatality in sheep and goats due to peste des
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petits ruminants Breeds
Total
*Morbidity
Animals
N
**Mortality
%
N
%
***Case Fatality (%)
Sheep breeds 472
33
6.99
7
Kajli
747
563
75.37
245
Dorper vs Kajli sheep
P Value = 0.001
P Value = 0.001
P Value = 0.083
5.01
Beetal
725
588
81.10
12
2.23
44.44
270
37.24
45.92
χ2 Value = 148.116
χ2 Value = 0.008
P Value = 0.001
P Value = 0.001
P Value = 0.927
Sheep
1219
596
Goats
1264
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χ2 Value = 287.594
48.89
252
20.67
42.28
48.65
282
22.31
45.85
χ2 Value = 0.005
χ2 Value = 0.637
χ2 Value = 0.608
P Value = 0.945
P Value = 0.425
P Value = 0.436
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2483
43.52
χ2 Value = 3.000
539
Total/Overall
32.80
χ2 Value = 122.853
Boer
Sheep vs Goats
21.21
χ2 Value = 219.485
Goat breeds
Boer vs Beetal goats
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48.77
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21.50
44.10
Data analysis by Chi-square test and df in each case was 1.
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Number of animals became sick
*Morbidity % =
Total number of animals at risk
X 100
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Number of animals died
**Mortality % =
***Case Fatality (%) = 672
Total number of animals at risk
X 100
Number of animals died Number of animals became sick
X 100
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Captions of Figures
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Fig. 1:
Photographs of intestines of a) a sheep and b) a goat died of peste des petits
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ruminants showing streaks of hemorrhages (zebra stripping) and blood clots
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(arrows). Fig. 2:
Photomicrographs of intestines of a goat (a and b) died of peste des petits
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ruminants, showing stunting and blunting of villi (arrows) with necrosis of
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tips of the villi, as well as sloughing of the villi (arrow head). There is
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infiltration of the lamina propria with mononuclear cells and a few
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neutrophil leukocytes (asterisk) along with diffuse edema of the submucosa
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and proliferation of fibrocytes, forming the submucosa much thickened
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(double-head arrow). H and E Stain. X 100. Fig. 3:
Photomicrographs of spleen of a sheep (a and b) died of peste des petits
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ruminants, showing marked depletion of lymphocytes in the white pulp,
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together with reticuloendothelial cell hyperplasia (arrow heads) and
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congestion (arrow). Dilated sinusoidal spaces are filled with macrophages
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and plasma cells, with a few neutrophils. H and E Stain. X 100. Fig. 4:
Photomicrograph of liver (a) of a goat died of peste des petits ruminants,
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showing congestion (arrow head), cellular infiltration (arrows) and many
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hepatocytes have under gone degeneration. b-d) kidneys of sheep and goat
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died of peste des petits ruminants showing renal tubular necrosis (arrows),
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detachment of tubular epithelium from the basement membrane and pooled
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in the lumen (arrow heads) and filling of renal tubules with proteinaceous
696
material and casts (asterisks). H and E Stain. a) X 40; b & d) X 200 and c)
698 699 700
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X400.
Fig. 5:
Photograph of trachea of a sheep died of peste des petits ruminants, showing a) severe congestion and b) severe congestion with froth.
Fig. 6:
Photographs of lungs of a goat died of peste des petits ruminants showing
701
severe congestion of apical lobe of the lungs (a-c: arrows) and deposition of
702
fibrinous materials (d: arrow heads).
703
Fig. 7:
Photomicrograph of lungs of a sheep died of peste des petits ruminants
704
showing a) alveoli filled with serofibrinous edema fluid mixed with
705
fibrinous exudate (asterisks), numerous alveolar macrophages and moderate
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thickened interalveolar septa (arrows) and extensive infiltration of
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mononuclear cells in alveoli (arrow heads); c & d) characteristic interstitial
709
pneumonia with extensive infiltration of erythrocytes, mononuclear cells and
710
increased histiocyte proliferation. H and E Stain. a-c) X 100 and d) X 400.
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Fig. 8:
Photomicrographs of lungs of a goat died of peste des petits ruminants showing lesions of bronchopneumonia. a-b) alveoli filled with serofibrinous
713
edema fluid mixed with fibrinous exudate (asterisks), congestion (arrows),
714
numerous alveolar macrophages and moderate numbers of neutrophil
715
leukocytes in alveoli; and intranuclear eosinophilic inclusions in alveolar
716
macrophages (arrows). H and E Stain. X400. Fig. 9:
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Photograph of Gel electrophoresis for peste des petits ruminants one-step RT-PCR using NP3 and NP4 primers (product size = 352 bp). Lane 1 =
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Ladder (bp); Lane 2 = Spleen sample; Lane 3 = Negative control; Lane 4 =
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Lungs sample; Lane 5 = Mesenteric lymph node sample; Lane 6 = Brachial
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lymph node sample; Lane 7 = Liver sample and Lane 8 = Positive control.
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Highlights of the manuscript Pathophysiology of Peste Des Petits Ruminants in Sheep (Dorper & Kajli) and Goats (Boer & Beetal) •
Peste des petits ruminants was studied in local (Kajli sheep; Beetal goats) as well as
•
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imported small ruminant breeds (Dorper sheep; Australian Boer goat).
Morbidity and mortality rates were significantly higher in indigenous Kajli sheep and Beetal goats as compared to Dorper sheep and Australian Boer goat.
•
On postmortem examination, lungs were congested and pneumonic, with nodular and
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cystic appearance and intestines were hemorrhagic with zebra stripping.
Histopathological lesions of respiratory tract were severe and characteristic of broncho
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interstitial pneumonia, bronchopneumonia, interstitial pneumonia and fibrinous pneumonia.
One-Step RT-PCR using NP3 and NP4 primers confirmed a PPR virus of 352 bp size in
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various organs.
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