Evidence for a relationship between Leishmania load and clinical manifestations Laura Manna a, * , Stefano Reale b , Fabrizio Vitale b , Angelo Elio Gravino a a Dipartimento di Scienze Cliniche Veterinarie, Università di Napoli Federico II, Via F. Delpino, 1-80137 Napoli, Italy b Istituto Zooprofilattico Sperimentale della Sicilia, Via R. Dicillo, 9-90139 Palermo, Italy article info Article history: Accepted 19 December 2008 Keywords: Clinical score Dog Leishmania infantum Real-time PCR abstract Visceral leishmaniasis (VL) is a life-threatening disease of medical, social and economic importance in endemic areas. Dogs are the main reservoir of Leishmania infantum. In this study, the authors investigated a group of 56 natural infected dogs to establish the relationship between parasite load and various clin- ical forms of leishmaniasis. The sick dogs were monitored at the beginning from clinical and physiological point of view. Leish- mania load was measured by real-time PCR assay on whole blood samples and lymph node aspirates, col- lected at the time of diagnosis. Our results indicate that a higher quantity of Leishmania DNA was found in the lymph nodes of dogs characterized by maximum clinical score. This interesting finding indicates the presence of a positive relationship between Leishmania load and clinical manifestations in dogs showing a severe clinical form of leishmaniasis. Ó 2009 Elsevier Ltd. All rights reserved. Leishmaniasis is an anthropozoonosis caused by a protozoan belonging to the genus Leishmania ( Herwaldt, 1999). Actually, there are 12 million of Leishmaniasis cases including cutaneous, muco- cutaneous and visceral forms. The annual incidence of VL is 500,000 (World Health Organization, 2001). Leishmaniasis is a severe disease in dogs, which may result in death without an opportune pharmacological treatment. The dogs are the main reservoir of L. infantum parasites and they play a cen- tral role in the transmission of the disease to humans by phlebot- omine sandflies (Ashford, 2000). Therefore, the use of a vaccine to control the disease in dogs may decrease the infection pressure of L. infantum for humans. Several tests have already been developed, in order to improve the control of leishmaniasis by efficient and reliable methods of diagnosis. Taking into account that the avail- able serological techniques based on anti-Leishmania antibodies detection are not entirely satisfactory because they do not discrim- inate between disease and asymptomatic infection (Sarkari et al., 2005), a real-time PCR assay was recently introduced to detect, quantitate and/or differentiate Leishmania organisms (Francino et al., 2006; Mary et al., 2004; Rolao et al., 2004; Schulz et al., 2003; Van der Meide et al., 2005). In a previous follow-up study on leish- maniotic dogs (Manna et al., 2004), the monitoring was done for 2 years after the end of the treatment. The authors demonstrated that the animals showing an apparently healthy state were PCR positive in skin and lymph node. However, it was recently demon- strated that the simultaneous quantitative evaluation of Leishmania DNA and cytokines by real-time PCR assay, allowed to predict the development of disease in asymptomatic infected dogs (Manna et al., 2006). Because few data are available on the Leish- mania load associated with different clinical forms of disease, the aim of this study was to investigate the relationship between par- asite burden and clinical sign scoring in different cases of leishmaniasis. The study included 56 sick dogs of different ages and breeds pre- sented at the Department of Veterinary Clinical Science of the Uni- versity Federico II of Naples, and diagnosed by serology (IFAT) and conventional PCR, as affected by canine leishmaniasis (Manna et al., 2004). The clinical assessment was performed in the dogs by observing the severity of signs attributable to Leishmania infection; animals were scored for clinical and for IFAT titre of anti-Leishmania antibodies on a scale from 0 to 10. The scored parameters were size of lymph nodes (0: normal; 1: enlargement); skin involvement (0: normal; 1: slight scaling and/or alopecia; 2: severe alopecia and/ or lesions); weight loss (0: absence; 1: moderate; 2: severe); haem- orrhagic diarrhea (0: absence; 1: presence); hyperthermia (0: ab- sence; 1: presence); ocular lesions (0: absence; 1: presence); orchitis (0: absence; 1: presence); haematuria (0: absence; 1: pres- ence); proteinuria (0: absence; 1: presence). The maximum score (10) was assigned to three dogs with renal failure and several bone lesions, while the scores of 1, 2 and 3 were assigned to IFAT titres of 1/40, from 1/80 to 1/160 and >1/160, respectively. With the consent of the dog owners, whole blood samples and lymph node aspirates were collected for real-time PCR assay. Whole blood and lymph node aspirates taken from three unin- fected dogs living in the area of Abruzzo region (Italy), where the 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.12.009 * Corresponding author. Tel.: +39 081 2536011; fax: +39 081 2536008. E-mail address: laumanna@unina.it (L. Manna). Research in Veterinary Science 87 (2009) 76–78 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc