Available online at www.sciencedirect.com

www.elsevier.com/locate/tvjl

The Veterinary Journal 174 (2007) 655–658

TheVeterinary Journal

Short Communication

Diagnosis of clinical cases of the nervous form of Maedi-Visnain 4- and 6-month-old lambs

Julio Benavides, Carlos Garcıa-Pariente, M. Carmen Ferreras, Miguel Fuertes,J. Francisco Garcıa-Marın, Valentın Perez *

Departamento de Patologıa Animal, Medicina Animal (Anatomıa Patologica), Facultad de Veterinaria, Universidad de Leon,

Campus de Vegazana s/n, 24071 Leon, Spain

Accepted 17 October 2006

Abstract

The nervous form of Maedi-Visna (MV) infection was diagnosed in four lambs aged 4 and 6 months, belonging to three differentAssaf flocks that were managed intensively for milk and meat production. The animals presented with hindleg ataxia that rapidly pro-gressed to complete recumbency. Lesions consisted of a moderate to severe non-purulent encephalitis affecting mainly the cerebellarpeduncles. MV virus was demonstrated in the damaged tissues by immunohistochemistry and polymerase chain reaction. The investiga-tion demonstrated that the clinical presentation of the nervous form of MV which is reported to occur in adult sheep can also be observedin young animals.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Maedi-Visna; Lentivirus; Nervous; Sheep

Maedi-Visna (MV) is a multisystemic chronic infectionof sheep that can affect the lungs, mammary gland, centralnervous system and joints (Dawson, 1980) with a lymphoidproliferation and mononuclear interstitial infiltration. It iscaused by MV virus (MVV), a retrovirus belonging to thelentiviral subfamily.

The nervous form, or visna, was first described in Ice-land as a chronic progressive paralytic disease of adultsheep (Sigurdsson et al., 1957). It is characterized by anon-suppurative demyelinating encephalitis that affectsthe white matter, composed of an infiltrate of lymphocyteswith macrophages and glial cells in the brain parenchyma,together with mononuclear perivascular cuffs (Sigurdssonet al., 1957, 1962). Other than the Icelandic epidemics,visna has been reported only rarely, usually accompanyingthe respiratory form (Palsson, 1990; Watt et al., 1992; Prit-chard et al., 1995) and always in sheep older than twoyears. However, in the Castilla y Leon region of Spain,

1090-0233/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tvjl.2006.10.014

* Corresponding author. Tel.: +34 987 291327; fax: +34 987 291103.E-mail address: dmavpp@unileon.es (V. Perez).

numerous cases of visna have been recently diagnosed,mostly in the Assaf breed (Benavides et al., 2006).

In caprine arthritis-encephalitis virus (CAEV) infection,a disease of goats similar to MV and caused by a lentivirusclosely related to MVV, an encephalomyelitis similar tothat of visna appears in adult animals although it can alsooccur in kids aged 2–6 months (Callan and Van Metre,2004). The objective of the present study was to describefour clinical cases of visna in Assaf sheep aged 4 and 6months, to characterize the lesions and to demonstratethe presence of MVV by immunohistochemistry (IHC)and polymerase chain reaction (PCR) methods.

Four sheep, aged 4 (n = 3) and 6 (n = 1) months pre-sented with rapid onset locomotor disorders that were con-sidered likely to be a nervous disease. The earliest sign wasa stumbling gait due to hindleg weakness and ataxia(Fig. 1). The animals were alert and ate when helped togain access to food. The status of the animals worsenedand their condition progressed over 4–6 days to completerecumbency with rhythmic movements of the four legs.No response to sonorous or visual stimulus could be

Fig. 1. A four-month-old lamb with visna, showing weakness of thehindlimbs and abnormal stance. The animal is leaning against the wall andis unable to remain upright.

Fig. 2. Severe encephalitis located in the cerebellar peduncles and thedeeper areas of the cerebellum. Perivascular cuffs, demyelination and adiffuse inflammatory infiltrate can be seen. H and E, Bar = 1 mm.

656 J. Benavides et al. / The Veterinary Journal 174 (2007) 655–658

detected at this time. The animals were afebrile, even whenrecumbent. No dyspnoea or respiratory signs were reportedin any case and no loss of weight was observed before andduring the onset of the clinical signs.

The animals were of the Assaf breed and although theycame from three different flocks located in the same areathey were unrelated. The flocks were managed intensivelyfor milk and meat production, had a previous history ofclinical cases of visna in adult sheep and had recordedMV seroprevalence rates >60% (Innotest, Hypen Biomed).

Euthanasia by intravenous injection of barbiturate fol-lowed by exsanguination was carried out in the affectedanimals, a necropsy was performed, and the brain andentire spinal cord were removed. After gross examinationby coronal sectioning, samples for histology were obtainedfrom six levels of the encephalon and the spinal cord. Theremainder of the organs were grossly examined, includingthe target sites for MVV (lungs, mammary gland andjoints) and samples from the lungs and the mammary gland(two animals) were taken for histological evaluation, aswell as from other organs.

The tissues were fixed in 10% buffered neutral formalin,dehydrated through graded alcohols and embedded in par-affin wax. Several sections (4 lm) were cut from each sam-ple and stained with haematoxylin and eosin (H and E).Immunohistochemical demonstration of MVV antigens inthe tissues was carried out by the avidin-biotin-peroxidasecomplex (ABC) method with 3,3 0diaminobenzidine aschromogen substrate, and with antibodies against MVVp27 and gp 130 proteins (Gelmetti et al., 2000; Benavideset al., 2006). Demonstration of MV provirus in paraffinwax-embedded tissue samples was performed by long ter-minal repeat (LTR)-PCR, as previously described (Bena-vides et al., 2006). A sample was considered positivewhen a 291-bp DNA fragment of the LTR sequence ofMVV provirus was obtained.

No gross changes were detected in the brain. Microscop-ically, the four animals showed lesions consistent with thenervous form of MV, characterized by a non-suppurativeencephalitis that was always seen in the cerebellar pedun-cles (Fig. 2) and extended to the deep cerebellar nucleiand the cerebellar cortex. Inflammatory changes alsoappeared in the medulla oblongata, mesencephalon andhippocampus, showing less severity. Encephalitis was mod-erate to severe and consisted of multifocal to coalescentfoci of inflammation formed by perivascular cuffing ofmononuclear cells and an infiltrate of macrophages, lym-phocytes and glial cells, located in the neuroparenchymasurrounding the cuffs (Fig. 3a). Areas of demyelinationand malacia were also present. No other significant grossor microscopic lesions were detected.

The presence of MVV antigens was demonstrated byIHC in tissues from each of the four animals (Fig. 3b). Apositive reaction was taken to be a fine granular brownishdeposit in the cytoplasm of cells consistent with macro-phages present in the areas of inflammation. MVV proviralDNA has been also demonstrated in tissue samples fromthe pons and cerebellar peduncles of the four sheep byLTR-PCR.

The most striking finding was the occurrence of clinicalcases of visna in four lambs aged 4 and 6 months of age. Toour knowledge, natural cases of the nervous form of MVhave only been diagnosed in sheep older than two years(Sigurdsson et al., 1962; Pritchard et al., 1995). Only in a

Fig. 3. (a) Perivascular cuffs formed mainly by macrophages andlymphocytes in an area showing demyelination and diffuse mononuclearinflammatory infiltrate. H and E, Bar = 100 lm. (b) Positively stained cellsconsistent with macrophages in an area of inflammation. IHC.Bar = 50 lm.

J. Benavides et al. / The Veterinary Journal 174 (2007) 655–658 657

previous study carried out in our department, 20/71 casesof visna were in sheep aged between 8 and 24 months(Benavides et al., 2006).

Microscopic lesions encountered in the four casesreported here were consistent with the nervous form ofMV and similar to those observed in natural cases in adultsheep (Sigurdsson et al., 1957, 1962; Watt et al., 1992). Thisfinding supports the previously suggested hypothesis(Georgsson et al., 1978) that the age of the animals doesnot influence the histopathological features of the visnalesions.

Other infectious causes of encephalitis in small rumi-nants such as listeriosis, louping-ill, borna disease or bacte-rial meningoencephalitis as well as degenerativeneuropathies as focal symmetrical encephalomalacia orpoliencephalomalacia were discarded on the basis of theclinical signs and gross and microscopic lesions (Summerset al., 1995).

In each of the cases described here the clinical signs pro-gressed rapidly to recumbency, in contrast with reports ofnatural cases in adult sheep that show a more prolongedduration of clinical signs (Dawson, 1980; Watt et al.,1992). This fact together with the presentation of the dis-ease in young animals, might suggest the implication ofneutropic and highly neurovirulent MVV strains. In othercases of lentiviral encephalitis (O’Neil et al., 2004) it hasbeen claimed that progression of the disease is related tothe viral load and initial host response to infection. Thisalso occurs in MVV infection (Dawson, 1980; Houwers,1990) and could have occurred in our animals if theyhad been infected as young lambs but were unable tomount defensive responses thereby allowing the virus toreach the encephalon. On the other hand, all four casescame from flocks managed intensively with the animals liv-ing indoors all the time and a high MV seroprevalence and

frequent clinical cases. From this it can be suspected thatthere was a high environmental viral load. Breed suscepti-bility, proposed as a factor that can influence the develop-ment of MV (Houwers, 1990), should be also consideredsince all the four cases belonged to the Assaf breed.

In CAEV infection, neurological clinical cases do occurin young kids (Callan and Van Metre, 2004). Moreover,natural transmission of CAEV from goats to sheep doesoccur (Shah et al., 2004) and cannot therefore be excludedin this report because the serological and PCR techniquesemployed were not specific for a particular small ruminantlentivirus. However, this possibility seems unlikely in viewof the lack of articular involvement, which is a typical fea-ture of CAEV infection (Callan and Van Metre, 2004),and the absence of known contact with goats. On the otherhand, MVV and CAEV are considered to be closely related(Callan and Van Metre, 2004) and the clinical presentationof visna in young animals could possibly be a new feature ofMV disease, as with CAEV infection in goats.

Further investigations focusing on the viral strains aswell as environmental and host factors in cases such asthese are needed. In the meantime, MVV infection shouldbe taken into account as a possible cause of neurologicaldisorders in lambs.

Acknowledgements

The authors wish to thank Javier Otaola and EmilioHerrera who collaborated by supplying the animals andDr. D. Gelmetti for the kind gift of antibodies. This workwas supported by grant LE52/04 from Junta de Castilla yLeon.

References

Benavides, J., Gomez, N., Gelmetti, D., Ferreras, M.C., Garcıa-Pariente,C., Fuertes, M., Garcıa-Marın, J.F., Perez, V., 2006. Diagnosis of thenervous form of maedi-visna infection with a high frequency in sheepin Castilla y Leon, Spain. Veterinary Record 158, 230–235.

Callan, R.J., Van Metre, D.C., 2004. Viral diseases of the ruminantnervous system. Veterinary Clinics of North America: Food AnimalPractice 20, 107–116.

Dawson, M., 1980. Maedi/visna: a review. Veterinary Record 106, 212–216.Gelmetti, D., Gibelli, L., Brocchi, E., Cammarata, G., 2000. Using a panel

of monoclonal antibodies to detect maedi virus (MV) in chronicpulmonary distress of sheep. Journal of Virological Methods 88, 9–14.

Georgsson, G., Petursson, G., Miller, A., Nathanson, N., Palsson, P.A.,1978. Experimental visna in fœtal Icelandic sheep. Journal of Com-parative Pathology 88, 597–605.

Houwers, D.J., 1990. Economic importance, epidemiology and control.In: Petursson, G., Hoff-Jørgensen, R. (Eds.), Maedi-Visna and RelatedDiseases. Kluwer Academic Publishers, Boston, pp. 83–117.

O’Neil, S.P., Suwyn, C., Anderson, D.C., Niedziela, G., Bradley, J.,Novembre, F.J., Herndon, J.G., McClure, H.M., 2004. Correlation ofacute humoral response with brain virus burden and survival time inpig-tailed macaques infected with the neurovirulent simian immuno-deficiency virus SIVsmmFGb. American Journal of Pathology 164,1157–1172.

Palsson, P.A., 1990. Maedi-Visna. History and clinical description. In:Petursson, G., Hoff-Jørgensen, R. (Eds.), Maedi-Visna and RelatedDiseases. Kluwer Academic Publishers., Boston, pp. 3–17.

658 J. Benavides et al. / The Veterinary Journal 174 (2007) 655–658

Pritchard, G.C., Done, S.H., Dawson, M., 1995. Multiple cases of maediand visna in a flock in East Anglia. Veterinary Record 154, 94.

Shah, C., Huder, J.B., Boni, J., Schonmann, M., Muhlherr, J., Lutz, H.,Schupbach, J., 2004. Direct evidence for natural transmission of small-ruminant lentiviruses of subtype A4 from goats to sheep and viceversa. Journal of Virology 78, 7518–7522.

Sigurdsson, B., Palsson, P.A., Grımsson, H., 1957. Visna, a demyelinatingtransmissible disease of sheep. Journal of Neuropathology 39, 519–528.

Sigurdsson, B., Palsson, P.A., Van Bogaert, L., 1962. Pathology of visna.Transmisible demyelinating disease of sheep in Iceland. Acta Neuro-pathologica 1, 343–362.

Summers, B.A., Cummings, J.F., de Lahunta, A., 1995. In: Summers,B.A., Cummings, J.F., de Lahunta, A. (Eds.), Veterinary Neuropa-thology. Mosby-Year Book, St. Louis, Missouri, USA.

Watt, N.J., King, T.J., Collie, D., McIntyre, N., Sargan, D., McConell, I.,1992. Clinicopathological investigation of primary uncomplicatedmaedi-visna virus infection. Veterinary Record 131, 455–461.