**5. Response to** *Anaplasma ovis* **infection in experimentally infected sheep**

Animal well-being has become a significant concern among consumers who expect food animals to be well treated, raised in idyllic environments, and free of disease. Consumers also expect their meat products to be free of residual antibiotics and therapeutic drugs. For these reasons, new approaches or alternatives to addressing animal diseases are needed. One approach is genetic selection for animals resistant to disease, that is: an approach whose focus is on accepting certain constraints of the environment and using breeds that can cope with these constraints, as opposed to the earlier approach which focussed on changing the environment to create opportunities for exotic breeds to be productive. But identifying the phenotype for disease resistance is difficult.

Tolerance to Tick-Borne Diseases in Sheep:

**5.1.1 Search for carriers and parasites** 

the HBBB allele at the beta globin loci.

**5.1 Materials and methods** 

**5.1.2 Animals** 

Highlights of a Twenty-Year Experience in a Mediterranean Environment 465

Thus, a project was set up to evaluate the response to anaplasmosis in susceptible and tolerant sheep breeds including the use of haemoglobin genetic systems as genetical markers

Sixty ewes were sampled from a flock extensively reared in the countryside near Bari. The flock consisted of approximately 250 heterogeneous subjects belonging mainly to TBD tolerant breeds and crossbreds. The presence of TBPs was checked in the blood samples by a PCR-based molecular approach as described above. Most of the sampled animals were found to carry *A. ovis* mixed with other TBPs. Based on the results of the flock survey and the consent given by the breeder, three ewes carrying *A. ovis* and *T. ovis* were selected and purchased. Following the experimental design (table 7) Lina and Zoppina were

Selected animals 7/8 months of age were involved in this study. Lambs less than six months of age were purchased and housed at the Medical Clinics of the Faculty of Veterinary Medicine of the University of Bary. Upon arrival at the Faculty of Veterinary Medicine, the animals were weighed and faecal samples were obtained to establish their worm burdens. Feet were checked for foot rot. The animals were dewormed with a broad spectrum anthelmintic. All of them were then housed in a tick proof isolation unit. In particular, in 2009 the lambs were

Fig. 5. Alpha-globin gene haplotypes detected so far in sheep, namely: haplotypes 1, 2 and 3 are normally duplicated (NH); haplotypes 4, 5 and 6 show extranumeral alpha gene arrangements (EH); particularly haplotypes 4 and 5 are triplicated while haplotype 6 is quadruplicated.

All the lambs were characterized by a normal duplicate alpha gene arrangement (Fig. 5) and most of them by homozigosity at the beta globin loci. Owing to high frequency of HBBA gene in the Suffolk breed, three out of the eight Suffolk lambs were HBBAB heterozygotes. In 2010 eighteen Altamurana lambs less than six months of age, housed and treated as above described, were selected based on different alpha globin genetic arrangements. Nine lambs were homozygotes for the normal duplicate alpha gene haplotype (NH), the others carrying an extra-numeral alpha haplotype (EH) (Fig.5); most of the 18 lambs were homozygotes for

selected based on different breed and equally divided between Suffolk and Comisana.

of tolerance to the disease. Summarized actions are described in table 7.

splenectomized in 2009 while Gilda in 2010, as described in subsection 3.4.

As to TBD, the response is known to be under multi-factorial regulation (Horin, 1998; Aguilar-Delfin et al., 2001). As highlighted in the above section 4, the phenomenon of tolerance is a broad-based one and possibly not unrelated to the erythropoietic system in different sheep breeds or to the haemoglobin genetic systems (Pieragostini et al., 2003; Pieragostini et al., 2006).

Anyway, the success of selection for disease resistance is dependent on correctly identifying the disease agent and the phenotype for disease resistance. For example, as to TBP in small ruminants, there are several reports concerning the presence of *Babesia, Theileria,* and *Anaplasma* species infecting sheep and goats in many countries world-wide but, in many regions of the Old and the New World, the identity of the tick-borne disease agents of sheep and goats and of their vector ticks is uncertain. But perhaps, the biggest challenge of selecting for disease resistance is to accurately identify the phenotype for disease resistance and/or to have reliable genetic markers with high predictive values for a disease phenotype. Phenotypic variability induced by parasites is a matter of fact, as impressively exemplified by the high number of haemoglobinopathies in human populations living in malariaendemic areas (Evans & Wellems, 2002).

Recalling Feynman's1 saying that nature repeats itself at every scale, we suggested that the unusual haemoglobin polymorphism recorded in Apulian native sheep breeds and the related functional effects might have an adaptive significance, also being somehow related to the selective pressure of tick borne parasites (TBP) (Pieragostini et al., 1994; Pieragostini et al., 2003; Pieragostini et al., 2006). Based on these considerations, we aimed to define the phenotype of the tick borne diseases in different sheep breeds starting from the one caused by *A. ovis*, the most common parasite in our area as confirmed by a small survey on sheep TBP performed in 10 farms (throughout Apulia) on 240 individuals. *A. ovis* was identified in 58% of samples, followed by *T. ovis* (5.8%) and *T. annulata* (4.5%). *Theileria* spp*.* were present in mixed infections with *A. ovis, B. ovis* (0.9 %) or *Babesia* spp. (0.9 %). In particular the presence of *A. ovis* was confirmed by specific polymerase chain reactions (PCRs) for *Anaplasma* spp. (Stuen et al., 2003) and *A. ovis* (de la Fuente et al., 2005; de la Fuente et al., 2007). Then PCRs followed by reverse line blot hybridization of the amplified 18SrRNA gene from *Theileria* and *Babesia* species, was used to detect specific probes for *Theileria/Babesia catch all, Theileria sp1 china, Theileria sp2 chinal, T. buffely, T. annulata, T. velifera, T. taurotragi, T. mutans, T. lestoquardi, T. ovis, B. bovis, B. bigemina, B. crassa, B. motasi, B. ovis, B. major, B. divergens, T. hirci, B. sp1 (Turchey), B. sp2 (Lintan*) (Schnittger et al., 2004).


Table 7. Experimental design.

 1 Richard Phillips Feynman (May 11, 1918 – February 15, 1988) was an American physicist who received the Nobel Prize in Physics in 1965.

Thus, a project was set up to evaluate the response to anaplasmosis in susceptible and tolerant sheep breeds including the use of haemoglobin genetic systems as genetical markers of tolerance to the disease. Summarized actions are described in table 7.
