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Keywords:

  • sheep-associated malignant catarrhal fever;
  • beef;
  • west bank

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

An outbreak of suspected malignant catarrhal fever (MCF) was investigated by molecular and histopathological assays. Of the 70 Holstein beef calf herds, 14 were affected by multiple clinical signs suggestive of MCF infection. These beef calves were housed next to sheep flocks. In the complete blood count, the 14 affected calves had severe anaemia with leucopaenia, lymphopaenia and neutropaenia. Upon PCR amplification using a hemi-nested PCR assay for the detection of the Ovine herpesvirus 2 (OvHV-2), bovine tissue samples from the mesenteric lymph nodes and spleen and ovine blood samples were shown to be positive with the expected PCR bands amplified. Direct sequencing of the hemi-nested PCR product confirmed the identity of the causative virus as OvHV-2. The histopathological findings confirmed the clinical and laboratory diagnosis of MCF. Collective clinical, PCR and histopathological data confirmed the identity of this outbreak to be a sheep-associated malignant catarrhal fever (SA-MCF).


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Malignant catarrhal fever (MCF) is a systemic disease of cattle caused by multiple viruses belonging to the new genus Macavirus (previously known as Rhadinovirus) of the subfamily gammaherpesvirinae (family herpesviridae) (Li et al., 2005; Davison et al., 2009). At least ten viruses are found in this genus (Crawford et al., 2002; Li et al., 2005), including the Alcelaphine herpesvirus 1 (AlHV-1) and Ovine herpesvirus 2 (OvHV-2). These viruses are the causative agents of both wildebeest (or African)-associated MCF and sheep-associated MCF, respectively (Plowright et al., 1960; McGeoch et al., 2005). A complete genome sequence of both viruses (AlHV-1, OvHV-2) has been published, with the genomes consisting of 130 608 and 135 621 bp, respectively (Ensser et al., 1997; Taus et al., 2007; Meier-Trummer et al., 2009).

Acute MCF in cattle caused by either OvHV-2 or AlHV-1 is very similar, although almost all cases of MCF in American cattle are caused by OvHV-2 (O'Toole et al., 1997). High fever and mucopurulent nasal discharges are very common along with other clinical signs, such as erosions, lameness and corneal opacity (Reid et al., 1986). The microscopic lesions include necrosis of the alimentary, urinary and upper respiratory epithelium, and vasculitis and perivascular cuffing. As a herpesvirus, OvHV-2 maintains a persistent infection in its hosts (sheep) through the process of latency (O'Toole et al., 1997). The persistent infection is present without causing clinically significant damage to the carrier animal. The virus reactivates into the lytic mode, during which infectious viruses are generated and transmitted to induce disease in cattle. Lymphocytes are known to be the host cells for OvHV-2 in sheep (Baxter et al., 1997). The primary shedding route of OvHV-2 is through nasal secretions (Li et al., 2004), although high levels of OvHV-2 DNA were found in the semen of rams, suggesting an important role for sexual transmission (Hussy et al., 2002).

This study describes a malignant catarrhal fever disease outbreak in a private beef cattle holding in Tulkarem (a city in the west bank), with characterization of the observed clinical signs, the histopathological findings and molecular characterization of the causative agent (OvHV-2). After this outbreak, we noticed the appearance of MCFV infection in sporadic cattle cases in neighbouring Jordan for the first time.

Methods and Materials

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Case history and clinical signs

The disease started in a private beef cattle holding in Tulkarem (a city in the west bank). The infection appeared in three Holstein beef calves with an age of 6–7 months, with the primary clinical signs of weakness, hypersensitivity, diarrhoea, a high fever (41.5°C), open-mouthed breathing, salivation and subsequent bilateral corneal opacity with mucopurulent oculonasal discharge. The course of these clinical signs lasted for 10 days. Fourteen of 70 animals were then found to be affected. The entire herd was slaughtered. Three sheep flocks were next to the affected calves. The first flock (flock no. 7) comprised 270 rams between 6 and 8 months of age. This flock had some sheep showing ocular discharge, arthritis and high fever. The second flock (flock no. 8) had 350 rams aged over 7 months. The third flock (flock no. 9) had 120 ewes below 6 months of age. Ocular discharge was observed in one ewe. The infection started in December, and the farm is an open farm without protection against cold, wind and rain. Tissue samples (mesenteric, retropharyngeal and medistinal lymph nodes, spleen, cerebrum, small intestines (ileum), spleen and kidneys) were taken from the affected calves and stored at −20°C. Whole blood and serum samples were taken from the affected beef calves and from the sheep flocks from jugular vein for a complete blood count using the HA-22/20/veterinary haematology analyzer. Analysis for complete blood count was carried out immediately after collection. Whole blood for viral DNA extraction and serum samples were stored at −20°C.

Viral DNA extraction

DNA was extracted from the white blood cells of the buffy coat and tissues (mesenteric lymph nodes and spleen) using a commercially available, Wizard Genomic DNA Extraction Kit (Promega, Madison, WI, USA). Briefly, 100 μl of buffy coat was added to 900 μl of cell lysis solution and mixed by inversion and was then incubated for 10 min at room temperature. The mixture was then centrifuged at 13 500 g for 1 min, the supernatant was discarded and the pellet was vortexed. Three-hundred microlitre of nuclei lysis solution was added, and the sample was mixed by inversion, after which 100 μl of protein precipitating solution was added. The solution was vortexed for 20 s. The tubes were then centrifuged at 13 500 g for 3 min and the supernatant was transferred to a new tube containing 300 μl isopropanol. After mixing and centrifuging at 13 500 g for 1 min, the supernatant was discarded, and 300 μl of 70% ethanol was added. Tubes were centrifuged at 13 500 g for 1 min, following which the ethanol was carefully aspirated, and the tubes allowed to air dry for 15 min. Finally, 100 μl of DNA rehydration solution was added to the tube, and the samples were incubated at 65°C for 1 h. The resulting DNA extracts were stored at −20°C in preparation for polymerase chain reaction (PCR).

PCR amplification and sequencing

A sensitive and well-established hemi-nested PCR assay targeting a DNA fragment in ORF 75 of the OvHV-2 genome was used in this study. The hemi-nested PCR primers were the 556 primer (5′-AGTCTGGGGTATATGAATCCAGATGGCTCTC-3′), the 775 primer (5′-AAGATAAGCACCAGTTATGCATCTGATAAA-3′) and the 555 primer (5′-TTCTGGGGTAGTGGCGAGCGAAGGCTTC-3′) (Baxter et al., 1993). In the first step, primers 556 and 775 were used to get a PCR product of 422 bp. In the second step, primers 556 and 555 were used to obtain a PCR product of 238 bp. for the PCR reaction, 10 pmol of each primer, 12.5 μl of Go Green Master Mix solution (Promega), 2.5 μl of 0.5 mm MgCl2, 1 μg of the DNA sample and nuclease-free water to a final volume of 25 μl were added for each sample. Thermal cycling conditions for both steps were 5 min at 94°C and then 40 cycles of 94°C for 30 s (the annealing temperature for both steps was 60°C for 60 s) and 72°C for 90 s then a final extension step at 72°C for 10 min. Amplification was performed using a GenePro thermal cycler (Bioer, China). After amplification, 10 μl of the reaction mixture was electrophoresed on 1.5% agarose gel and visualized using an UV transilluminator. The 238-bp PCR products from the calf and sheep DNA samples were sequenced using an ABI Prism 310 genetic analyser at the Princess Haya Biotechnology Centre (Jordan University of Science and Technology, Jordan). Sequences were aligned using BioEdit (7.0.5.3) and MUSCLE (3.7) software (European Bioinformatics Institute, Cambridge, UK).

Histopathological examinations

Frozen cerebrum, small intestine (ileum) and kidney samples were received for histopathological examination. The frozen tissues were left at room temperature to thaw and were then fixed in 10% buffered formalin for 24 h. Tissues were processed routinely and embedded in paraffin wax. 4-μm-thick sections were cut and stained with the haematoxylin and eosin (H and E) stain and examined histopathologically.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Clinical signs of weakness, hypersensitivity, dry rough hair coat, diarrhoea, high fever, open-mouthed breathing and salivation and bilateral corneal opacity with mucopurulent oculonasal discharge were observed in sick calves (Fig. 1). Examination of blood samples from these calves showed severe leucopaenia, along with lymphocytopaenia, neutropaenia and anaemia (this might be due to purulent inflammation from a secondary bacterial infection and the presence of ectoparasites namely ticks), with values below the normal ranges in the complete blood count. An increase in the number of immature leucocytes was noticed. Six bovine blood DNA samples, two bovine mesenteric lymph nodes samples, one bovine spleen DNA samples and three ovine blood DNA samples were subjected to the hemi-nested PCR amplification. No PCR product was obtained for the bovine blood samples in the hemi-nested PCR. The ovine blood samples were positive for both steps of the hemi-nested PCR. The bovine tissue DNA samples (two mesenteric lymph nodes and one spleen) were positive for both steps of the hemi-nested PCR, showing the presence of both bands (422 and 238 bp) (Fig. 2). Two PCR products of 238 bp, one from a bovine mesenteric lymph nodes sample and the other from the ovine blood DNA sample, were sequenced by direct sequencing of the DNA fragment in ORF 75 of the OvHV-2 genome. The sequencing results confirmed the identity of both PCR products (bovine and ovine) as ovine herpes virus 2 (OvHV-2) fragments.

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Figure 1. Holestine calf with corneal opacity, mucopurulent oculonasal discharge and salivation.

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Figure 2. Agaroes gel elecrtophoresis of a hemi-nested PCR amplified specific OvHV-2 fragment. Panel A is the first step PCR (primers 556 and 775), panel B is the second step PCR (primers 556 and 555). Samples 1,2,3,4,5 and 6 are bovine blood DNA samples: all are negative for both steps. Bovine lymph nodes (L1 and L2) and spleen (S) samples show positive results for both steps (422 and 238 bp). Ovine blood DNA samples (7,8 and 9) show positive results for both steps.

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The histopathological examination was limited because of freezing artefacts. Throughout the examined cerebral section, the blood vessels were variably cuffed with mononuclear cells, primarily lymphocytes (Fig. 3). The ileum showed larger sized Peyer's patches with severe lymphocyte depletion and necrosis (Fig. 4). The kidney sections showed multifocal moderate non-suppurative interstitial nephritis (Fig. 5). Lymphocytes were the predominant cell type. The lymphocytes primarily infiltrated the perivascular and periglomelular areas. The walls of some of the blood vessels within the small intestine and kidney were necrotic and disrupted by lymphocytes (vasculitis). The perivascular tissues exhibited necrotic debris and moderate lymphocyte infiltrates (Fig. 6). A few thrombosed blood vessels were present (Fig. 6).

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Figure 3. Holstein calf Cerebrum. Multilayered mononuclear perivascular cuffs. The cerebrum exhibited freezing artifacts*. H&E. Bar = 50 μm. Inset shows that the lymphocytes are the primary mononuclear cells in the perivascular cuff. H&E. Bar = 10 μm.

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Figure 4. Holestine calf. Small intestine (ileum). Marked enlargment of the Payer's patches with severe lymphocyte depletion and necrosis. H&E. Bar = 50 um. Inset showes thrombosed submucosal blood vessel with perivascular lymphocytic inflitrate. H&E. Bar = 1 um.

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Figure 5. Holestine calf. Kidney. Moderate multifocal non-suppurative interstitial nephritis. H&E. Bar = 50 μm.

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Figure 6. Holestine calf. Blood vessels. The blood vessel wall is necrotic and disrupted by inflammatory cells (vasculitis). The perivascular tissues exhibited necrotic debris and moderate numbers of lymphocytes. H&E. Bar = 10 μm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The first documented outbreak of sheep-associated malignant catarrhal fever (SA-MCF) in the Middle East was reported in Israel, followed by an outbreak in Saudi Arabia (Brenner et al., 2002; Abu Elzein et al., 2003), with both outbreaks affecting calves with a similar epidemiological pattern. Outbreaks of SA-MCF have been reported in all continents: Asia (Wani et al., 2006), Australia and New Zealand (Tham, 1997; Tomkins et al., 1997), Africa (Mushi and Rurangirwa, 1981), Europe (Muluneh and Liebermann, 1992) and in North and South America (Berezowski et al., 2005; Costa et al., 2009). In this study, we characterize an outbreak of SA-MCF in Tulkarem (a city in the west bank) which we believe was the focal point for the spreading of SA-MCF to the neighbouring Jordan as we noticed later.

SA-MCF was observed in 14 of 70 calves in a Holstein beef herd of private holding in the city of Tulkarem in the west bank. This herd was adjacent to 3 sheep flocks in which some animals were showing similar clinical signs of malignant catarrhal fever, indicating that the SA-MCF started in these sheep flocks. The epidemiology and the transmission of SA-MCF from sheep to cattle is affected by the age of susceptible sheep [older than 2 months (Li et al., 1998)], which was the case in the observed outbreak, as the age of the sheep in all flocks was above 6 months.

The hemi-nested PCR used in this study is considered by many to be the best molecular PCR assay for the diagnosis of SA-MCF in clinical samples (Li et al., 1995; Muller-Doblies et al., 1998). Bovine blood samples were negative for the hemi-nested PCR, indicating that the viraemia stage was not present at the time of sample collection, but ovine blood samples were still showing the viral DNA in the PCR. On other hand, bovine tissue (mesenteric lymph nodes and spleen) were positive for the PCR assay, as the target cells for OvHV-2 infection is the CD8+ T lymphocytes (Simon et al., 2003). The involvement of lymphocytes was investigated by the experimental infection of rabbits with OvHV-2 (Anderson et al., 2007). Anderson et al. reported that OvHV-2 was found in the lymphoid infiltrates found in the appendix, mesenteric lymph nodes, spleen and the lymphoid cell accumulations in the liver and kidney. The same histopathological picture was seen in this study, with the blood vessels in the cerebrum being cuffed with lymphocytes. The ileum showed moderate-to-severe hyperplastic Peyer's patches with severe lymphocyte depletion and necrosis. The kidneys had multifocal moderate non-suppurative interstitial nephritis with lymphocytes being the majority of cells.

In this study, a sheep-associated malignant catarrhal fever (SA-MCF) outbreak in beef calves was characterized by a hemi-nested PCR coupled with sequencing, along with clinical and histopathological data.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors wish to thank Dr. Belal Yousef Abu-Helall and Dr. Ibraheem Mahmood Al-Zuheir for their technical assistance.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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