Sequential haematological and serum biochemical changes in Black Bengal goats infected with a local isolate of peste des petits ruminants virus from Bangladesh

Abstract Background Knowledge of sequential changes in haematobiochemical parameters of infected animals helps in the formulation of appropriate supportive therapy. Objective We investigated the sequential haematological and biochemical changes in peste des petits ruminants (PPR)‐infected Black Bengal goats. Methods Goats were either infected with PPR virus (PPRV; n = 8) or sham infected with sterile phosphate‐buffered saline (n = 4) via the intranasal route. Blood and sera were collected from both groups at different days post‐infection (dpi) and analysed. Goats were sacrificed at different dpi and the amount of PPRV RNA in different tissues was quantified by real‐time RT‐PCR. Results The PPRV‐infected goats showed mild depression and scanty nasal secretions starting at 4 dpi which became severe with high fever (106°F), dyspnoea, stomatitis, profuse orinasal discharge and diarrhoea at 9–13 dpi. PPRV RNA was detected in different tissues of infected goats. Severe lymphocytic leukopenia (at 18 dpi) was observed in infected goats. Total protein and albumin decreased in infected goats starting at 10 dpi. An elevated level of enzymes (alkaline phosphatase, creatine kinase, aspartate transaminase and alanine transaminase) and metabolites (blood urea nitrogen and urea B) were found in infected goats starting at 7–10 dpi, suggesting damages in the liver and kidneys. PPR‐infected goats showed elevated sodium and chloride ions starting at 7 dpi. The majority of infected goats were seroconverted by 14 dpi. Conclusions Anti‐diarrheal agents, aqua solutions and other medicine to support liver and kidney functions could be considered as supportive therapy against PPRV infection.


| INTRODUC TI ON
In Bangladesh, goat rearing has been practiced in both rural and urban areas for decades and the goat is considered as poor man's cow because of its immense contribution to the livelihood of rural people (Ahmed, 2017). The goat offers a supply of quality meat, milk and hide that contribute to the national economy. In the year 2017-2018, there were about 26.1 million goats in Bangladesh of which about 90% were Black Bengal goats (BBS, 2019). Extreme climatic conditions such as heavy rainfall and flood, lack of grazing lands, parasitic infestation and outbreaks of various infectious diseases hinder successful goat farming in Bangladesh (Ahmed, 2017).
Peste des petits ruminants (PPR), the number one killer of goats, is considered as one of the major constraints in sustainable goat farming in Bangladesh (Siddiky, 2013). The first outbreak of PPR in Bangladesh was reported in 1993 (Islam et al., 2001). Since then, outbreaks of PPR have regularly been reported across the country (Begum et al., 2018;Bhuiyan et al., 2014;Chowdhury et al., 2014;Rahman et al., 2011Rahman et al., , 2016. Provision of inadequate veterinary services combined with poor diagnostic facilities stalled the successful PPR control programme (Haider et al., 2017).
The prognosis of acute PPR is usually very poor, especially when aggravated with secondary bacterial infection, poor nutrition and other stresses (Saliki, 1988). Supportive therapy with fluid, electrolytes and antibiotics to prevent secondary bacterial infection can reduce the mortality of PPR Yousuf et al., 2015). Knowledge of changes in the haematobiochemical parameters of PPR-infected goats is necessary to formulate appropriate supportive treatment (Das et al., 2015). In addition, based on haematobiochemical changes at different stages of the disease, a different treatment plan will be necessary.
Several studies have been conducted to assess haematobiochemical alterations of goats died of natural PPR outbreaks and generated conflicting findings (Das et al., 2015;Sharma et al., 2012). The progressive changes in the haematobiochemical parameters of goats infected with PPR could not be evaluated on natural PPR infection. Therefore, the study was designed to assess the sequential haematological and serum biochemical alterations of Black Bengal goats experimentally infected with a Bangladeshi strain of PPRV.

| Animals
Twelve healthy Black Bengal goats aged between 4 and 8 months with a history of no vaccination against PPR were purchased from local markets. Animals were kept in relative isolation for 2 weeks with adequate feed and water. The goats were dewormed using subcutaneous 'Ivermectin' injection as per manufacturer's recommendation. All goats were tested for anti-PPRV antibodies with a commercial competitive ELISA kit (ID Screen PPR Competition, ID-Vet, Montpellier, France) and found seronegative for PPRV.

| Ethics statement
All applicable national and institutional guidelines for the care and use of animals were followed. The study was carried out in accordance with the recommendation of the

| Preparation of PPR virus inoculum
For experimental infection, PPR virus inoculum was prepared from a local isolate (BD/PPRV/2015/1; lineage IV) collected during a field outbreak in 2015. The virus was isolated in primary goat kidney cell culture. Briefly, a 20% tissue homogenate of lymph node was prepared from a PPR-infected goat using sterile PBS. A monolayer of primary goat kidney cells was then infected with 100 µl of the tis-

| Experimental infection and sample collection
Goats were divided into two groups: infected (n = 8) and uninfected control (n = 4) and housed separately. Goats from the infected group received 2 ml of the PPRV inoculum containing 5.6 log 10 TCID 50 /ml via the intranasal route. Control goats received 2 ml sterile PBS via the intranasal route. Goats from both groups were monitored twice daily for feed intake, clinical signs, body temperature and oral lesion development. Blood samples were collected at 0, 1, 2, 3, 5, 7, 10, 14, 18 and 21 days post-infection (dpi). After collection, 2 ml of blood was transferred to a sterile vial containing ethylenediaminetetraacetic acid (EDTA) @ 1 mg/ml blood for routine haematological examination. Five millilitre of blood was also transferred to another sterile tube without EDTA. After clotting of the blood, sera were collected and centrifuged at 900 g for 10 min. The supernatant was transferred to fresh tubes and stored at −20°C until use.

| Competitive enzyme-linked immunosorbent assay (ELISA)
A competitive ELISA kit (ID Screen PPR Competition, ID-Vet, Montpellier, France) was used to measure the amount of PPRVspecific antibodies in sera of PPRV-infected and healthy goats. As mentioned in the kit, a sample having a competition percentage (CP) value of ≤35 was considered as seropositive.

| Quantification of PPR virus load in different tissues
One PPRV-infected goat was euthanized at each time point of 5, 7, 14 and 18 dpi. Routine necropsy was performed and gross lesions were recorded. Tissues from pre-scapular lymph node, liver, kidney and spleen were collected for quantification of PPRV RNA in tissues.
To this end, a 20% tissue homogenate was prepared in sterile PBS.
Total RNA was extracted with PureLink RNA Mini Kit (ThermoFisher Scientific, USA) and quantified by NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, USA). For normalization and internal control, 20 ng of total RNA was used per reaction in all rtRT-PCR.

| Haematobiochemical analysis
Routine haematological examination of whole blood samples collected at different time points post-infection was performed by the standard method (Lamberg & Rothstein, 1978). Routine haematological parameters such as haemoglobin, erythrocyte sedimentation rate, packed cell volume, total erythrocyte count, total leucocyte count and differential leukocyte count were tested. In addition, different serum biochemical constituents such as total protein, albumin, glucose, bilirubin, blood urea nitrogen, creatine kinase, alkaline phosphatase, alanine transaminase and aspartate transaminase, inorganic phosphorus and calcium were analysed using an automated T80 Ultraviolet-visible spectroscopy (UV/VIS) spectrophotometer (PG Instruments, UK). Furthermore, an automated electrolyte analyser GENLYTE 3000A (IVD) was used to analyse serum electrolytes (sodium, potassium and chloride ions) using a commercial kit (Electrolyte solution, Biogen, GmbH, Germany).

| Statistical analysis
Statistical analysis was performed using the software package GraphPad Prism Version 5.0. One-tailed non-parametric Mann-Whitney U test was used to calculate the statistical differences in haematological and biochemical profiles between PPRV-infected and healthy goats.

| Host responses
The PPRV-infected goat showed mild depression and scanty nasal secretions starting at 4 dpi which was followed by high fever (104-105°F; Figure 1a) and watery oculonasal discharge for the next 3-5 dpi. Body temperature further increased (106°F) during 9-13 dpi with severe anorexia, dullness, dyspnoea, stomatitis, profuse orinasal and ocular discharge and diarrhoea. At 14 dpi, infected goats showed severe dyspnoea, profuse diarrhoea and orinasal inflammation with normal or sub-normal body temperature followed by death. Goats from the control group were healthy with normal body temperature throughout the study period. In this experiment, 3 of 8 goats died and the rest of the goats were sacrificed at different time intervals as mentioned earlier. Therefore, actual mortality was showed erosive necrosis at 10 dpi and the severity increased over time. The trachea was congested at 7 dpi and filled with white froth.
The lungs showed gradual consolidation and almost a major part of it was consolidated at advance stages. The enlarged and oedematous spleen was found in infected goats at 10 dpi, which became pale and atrophied at 14 dpi. White to greyish necrotic spot on the outer surface of the liver was found at 7 dpi. The kidneys in few infected goats were severely haemorrhagic and inflamed. On histopathology, the liver showed fatty change and single hepatocellular necrosis at an early stage which became multicellular at advance stage. The kidneys showed necrotic tubules, fusions of tubular epithelial cells and haemorrhages. The competitive ELISA of sera collected from PPRV-infected goats showed early seroconversion stating at 7 dpi in few animals (Figure 1b). Eventually all survived goats were seroconverted by 14 dpi (Figure 1b).

| Quantification of virus load in tissues
The for liver and spleen, respectively). Kidneys showed a moderate virus load throughout the study period with Ct value around 30.

| Sequential haematological changes in experimental PPRV-infected goats
The sequential changes in haematological parameters were analysed

| Serum biochemical analysis of experimental PPRV-infected goats
The progressive changes in serum biochemical parameters of PPRV-infected goats were analysed. Black Bengal goats infected with PPRV showed a significantly lower amount of total protein (hypoproteinemia) starting at 14 dpi ( Figure 3a)  The concentration of AST increased significantly at 18 dpi in infected goats compared withhealthy goats (Figure 4c). The concentration of another serum enzyme ALT also increased significantly starting at 7 dpi in infected goats (Figure 4d). Thus, PPR affected goats showed elevated serum enzymes.

| Analysis of serum electrolytes of experimental PPRV-infected goats
The progressive changes in serum electrolytes such as sodium, po-

| D ISCUSS I ON
Experimental PPR infection induced early fever in Black Bengal goats starting at 4 dpi which became severe during 9-13 dpi with dyspnoea, cheilitis, stomatitis and gingivitis leading to reduced feed intake. The significant rise in body temperature together with respiratory distress in PPR-infected goats were also found in several recent studies (Begum et al., 2018;Chowdhury et al., 2014;Truong et al., 2014;Ugochukwu et al., 2018).
However, during the advanced stage of the disease (>14 dpi), The majority of the study describing natural PPR outbreaks in goats showed severe anaemia attributed to massive haemorrhages in different visceral organs and disseminated intravascular clot (Begum et al., 2018;Das et al., 2015;Sahinduran et al., 2012;Sharma et al., 2012). However, we found a comparable number of total erythrocytes and haemoglobin concentrations in both infected and control goats throughout the experimental period. Such variation could be due to good management, routine deworming and good nutritional status of the animals in the present experimental study (Begum et al., 2018;Sharma et al., 2012). Few studies also reported an elevation in the amount of total erythrocyte number and haemoglobin concentration in goats died of natural PPR outbreaks which could be due to diarrhoea leading to dehydration and haemoconcentration Kataria et al., 2007). Analysis of leukocyte counts showed a significant decrease in the number of total leukocytes at 14 and 18 dpi in infected goats which was marked by lymphopenia at 18 dpi similar to what has been shown in natural PPR outbreaks (Begum et al., 2018;Islam et al., 2018;Kataria et al., 2007). Such reduction in leukocyte counts could be due to initial proliferation of the virus in lymphoid organs followed by destruction of the lymphocytes as the virus has an affinity to lymphocytes (Aruni et al., 1998), as well as bone marrow suppression (Obi & Oduye, 1985). In line with this, we found significant lymphoid destruction with high virus load in lymph nodes and spleen of PPR-infected goats. However, the number of neutrophils increased slightly in the PPR-infected goats at 18 dpi, suggesting secondary bacterial infections (Begum et al., 2018;Islam et al., 2018;Kataria et al., 2007).
These further support liver and kidney dysfunctions. Pathological investigation also revealed hepatocellular necrosis and hepatitis in the liver and tubular necrosis in the kidney starting at 7 dpi which mirrored the elevated serum enzymes and reduced protein level.
Moreover, a high virus load in the liver and kidney was also found in the infected goats. A similar phenomenon has also been recorded in Black Bengal goats died of natural PPR outbreaks (Begum et al., 2018). In line with two previous studies (Das et al., 2015;Kataria et al., 2007), we also found a significant increase in the concentration of sodium and chloride ions in PPR-infected goats at 14 dpi owing to severe diarrhoea and dehydration (Begum et al., 2018;Chowdhury et al., 2014).
In conclusion, PPR virus-infected goats died due to (1)  The funder had no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report and in the decision to submit the article for publication.

CO N FLI C T S O F I NTE R E S T
None declared.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.373.