**3.3 Piroplasmosis in naturally infected tolerant sheep**

Resistance is a dynamic process of parasite regulation by the host. The pathogen must penetrate host cell barriers in sufficient numbers, attack target cells and replicate. Sub-clinical or clinical expression of the disease is dependent on the pathogen's virulence and the interaction between pathogen and host characteristics. Particularly, the phenomenon of tolerance to tick borne pathogens (TBP) is closely linked to a particular type of anaemia which is generally the symptom *par excellence* of the disease. In the tolerant animals, as shown in a study carried out on Altamurana sheep, this takes a benign macrocytic and hyperchromic form. A comparison of the haematological parameters of healthy sheep with those of sick sheep in table 4 showed that the latter presented a numerical deficiency of red blood cells that was compensated by the fact that the mean corpuscular volume (MCV) increased by about 50% as did the mean corpuscular haemoglobin (MCH). The results shown in table 4 did not stem from a dedicated investigation because the haematologic alterations were met with by chance when investigating on the functional effect of a rare alpha globin gene variant. At sampling, the affected sheep did not show any patent signs of the disease and thus only the haemocromocytometric parameters, the related observation of blood films and the results of the osmotic fragility test led to classifying the sampled animals in healthy and affected. Observation of blood films in this study and in other subsequent occasional analyses on Apulian sheep in similar conditions, highlighted that in most cases there were mixed infections in which *Anplasma* spp. and/or *Theileria* spp. and/or *Babesia* spp. occurred at the same time (Fig. 3). *B. ovis* was consistently present in the blood films of the affected animals with visible symptoms of haematuria. This fact, taken together with the evidence from tests on Romanov sheep infected and killed by babesiosis, convinced us that *B. ovis* was one of the causes of the pathogenetic activity in Apulian sheep, and certainly in non-native breeds. However, diseases often occur in clusters of time (years, seasons, production cycles, etc.) and space (herd, pasture, farm, region, etc.) and the prevalence of this pathogen was never the target of a dedicated epidemiological investigation.

Fig. 3. Blood film showing a mixed infection of *Anaplasma* spp. and *Babesia ovis.* 

Resistance is a dynamic process of parasite regulation by the host. The pathogen must penetrate host cell barriers in sufficient numbers, attack target cells and replicate. Sub-clinical or clinical expression of the disease is dependent on the pathogen's virulence and the interaction between pathogen and host characteristics. Particularly, the phenomenon of tolerance to tick borne pathogens (TBP) is closely linked to a particular type of anaemia which is generally the symptom *par excellence* of the disease. In the tolerant animals, as shown in a study carried out on Altamurana sheep, this takes a benign macrocytic and hyperchromic form. A comparison of the haematological parameters of healthy sheep with those of sick sheep in table 4 showed that the latter presented a numerical deficiency of red blood cells that was compensated by the fact that the mean corpuscular volume (MCV) increased by about 50% as did the mean corpuscular haemoglobin (MCH). The results shown in table 4 did not stem from a dedicated investigation because the haematologic alterations were met with by chance when investigating on the functional effect of a rare alpha globin gene variant. At sampling, the affected sheep did not show any patent signs of the disease and thus only the haemocromocytometric parameters, the related observation of blood films and the results of the osmotic fragility test led to classifying the sampled animals in healthy and affected. Observation of blood films in this study and in other subsequent occasional analyses on Apulian sheep in similar conditions, highlighted that in most cases there were mixed infections in which *Anplasma* spp. and/or *Theileria* spp. and/or *Babesia* spp. occurred at the same time (Fig. 3). *B. ovis* was consistently present in the blood films of the affected animals with visible symptoms of haematuria. This fact, taken together with the evidence from tests on Romanov sheep infected and killed by babesiosis, convinced us that *B. ovis* was one of the causes of the pathogenetic activity in Apulian sheep, and certainly in non-native breeds. However, diseases often occur in clusters of time (years, seasons, production cycles, etc.) and space (herd, pasture, farm, region, etc.) and the prevalence of this pathogen was never the

**3.3 Piroplasmosis in naturally infected tolerant sheep** 

target of a dedicated epidemiological investigation.

Fig. 3. Blood film showing a mixed infection of *Anaplasma* spp. and *Babesia ovis.* 


Table 4. Comparison of the haematological parameters recorded in healthy and affected Altamurana sheep (mean values standard errors). Modified from Pieragostini & Petazzi, 1999. (RBC=Red Blood Cells; Hb=Haemoglobin %; PCV= Packed Cell Volume; MCV= Mean Corpuscular Volume; MCH=Mean Corpuscular Haemoglobin, MCHC=Mean Corpuscular Haemoglobin Content; WBC=White Blood Cells; MCF=Mean Corpuscolar Fragility) \*P<0.05;\*\*P<0.01; n.s.= not significant.

#### **3.4 Anaplasmosis in naturally infected splenectomized sheep**

Anaplasmosis is one of the most important tick-borne diseases of ruminants worldwide. The disease is caused by infection of animals with the obligate intraerythrocytic bacteria *Anaplasma* spp. which is classified in the family *Anaplasmataceae*, order *Rickettsiales* (Dumler et al, 2001). This section includes some experiences with sheep splenectomy and describes disease onset and course in eight splenectomized TBD-tolerant sheep that were naturally infected with piroplasms. Though the trials had been performed in different time periods, the results obtained were very similar and the facts surrounding the experiments gave us both general and specific insights into the field of splenectomy of carrier sheep from areas where endoerythrocytic parasites are endemic.

Particularly in the first trial, the surgical operation had two purposes: a) to evaluate the rôle of the spleen as a filter-pad to check parasites and as modulator of the direct response to anemia; b) to obtain a high number of parasites in the blood to prepare a local specific antigen.

The following trials were mainly related to the need to obtain *A. ovis* which was isolated from splenectomized sheep allowed to be naturally infected pasturing in tick areas.

Splenectomy was slightly traumatic for all the subjects and 24 hours after the surgical operation the sheep showed normal functions. The sheep were identified with female names for easier checking. Clinical evaluation was done on a daily basis and rectal temperatures were recorded every morning for 12 weeks post splenectomy. Blood and serum samples were routinely collected twice a week during the observation period. Haematological variables were evaluated using a haematology analyzer. The erythrocyte fragility test was performed by exposing erythrocytes to hypotonic saline solutions decreasing by 0.02% starting from 0.86%. Parasites in the blood were checked by Giemsa staining every 3 days. During the acute phase of the disease, the most important haematological values, erythrocyte fragility and parasitaemia were monitored daily. In the case of Gilda, Lina and Zoppina, which were part of the experiment to check the response to *A. ovis* infection of

Tolerance to Tick-Borne Diseases in Sheep:

0%

0%

0

5

11

17

23

28

32

35

37

**Days after splenectomy**

expressed as percentage of red blood cells parasitized in Lina and Zoppina, the two splenectomized sheep monitored over a five week period after splenectomy.

parasites; iii) disease relapse might be accounted for by the persistence of *A. ovis.* 

39

Fig. 4. Trend of PCV values and parasite densities (*An=Anaplasma ovis* and *Th=Theileria ovis*)

During the recovery period, clinical examination revealed only pale mucous membranes. The pattern of evolution of the disease and recovery in Gilda was almost the same as in Lina except for a higher level of parasitaemia. In this instance *A. ovis* was the sole agent of the illness of our sheep. Secondly, the increase in *A. ovis* is apparently slowed down by the presence of *T. ovis* which seems to have a *'*buffer effect*'* on the appearance of anaplasmosis but not in tempering its severity. Three points emerge from these results: i) in contrast to babesiosis and to the literature of some years ago (Radostits et al., 2000), hemoglobinuria did not occur in any of the seven severe cases of anaplasmosis; ii) treatment produced an immediate reduction in parasitaemia without leading to a complete clearance of the

Though not a novelty, in areas with enzootic erythrocytic parasitoses, even apparently healthy breeding animals may host pathogens and show tolerance and/or premunition to them without presenting with circulating parasites. This is a limit that should always be taken into account and may constitute a complication for any stress-associated situation. Anaemia secondary to anaplasmosis may evolve in a remarkably violent fashion probably due to the mechanism effected by the reticulo-endothelial system (RES) virtually with no

44

46

49

63

77

87

95

99

106

114

0

10

20

PCV (g/dl)

30

40

5%

**Parasitemia**

10%

15%

20%

0

8

17

25

32

38

**Days after splenectomy**

42

46

52

77

83

90

**Zoppina**

Th An PCV

94

101

114

PCV (g/dl)

10%

20%

**Parasitemia**

30%

40%

Highlights of a Twenty-Year Experience in a Mediterranean Environment 461

**Lina**

Th An PCV

different sheep breeds, described in section 5, parasite density was estimated on thin blood film and expressed as the percentage of parasitized red blood cells.

Fifteen days after the splenectomy, the general situation worsened and the animals became anorexic, staggering with a severe anaemia and dehydration. At the same time the RBC, Hb% and PCV values dropped (Tab. 5), and a number of organisms started appearing in the blood films (Rosalba showed a carpet of *A. ovis*; Stella a great deal of *A. ovis*; Lisa and Lola a great deal of *A. ovis* and a few *Babesia* spp.; Claretta a great number of *Theileria* spp.). Rosalba and Stella died of severe anaemia respectively 24 hours and 4 days after the diagnosis despite specific drugs and whole blood trasfusions with blood drawn from a donor subject. Claretta, Lisa and Lola showed less violent initial symptoms, the anaemic crisis was less severe and following a therapy with anti-protozoal drugs associated with desametazone they gradually began to eat and became clinically and haematologically healthy in 15-20 days. Since Lina, Zoppina, and Gilda, were included in the above cited experimental design to investigate the tolerance to *A. ovis*, they were constantly monitored and parasitaemia was recorded every two days after splenectomy. The cases of Lina and Zoppina allowed comparison between a mixed infection by *T. ovis* and *A. ovis* and an almost single infection by *A. ovis*. Interestingly, the two sheep coped differently with the infections. Though both animals were positive for *A. ovis* and *T. ovis* after splenectomy, the maximum of parasitized erythrocytes (MPE) by *T. ovis* peaked to 17% in Zoppina, while in Lina *T. ovis* caused a latent infection. Conversely, MPE by *A. ovis* in Zoppina was less than a half that of Lina (Tab. 5).


Table 5. Summary of clinical findings recorded in eight sheep splenectomised in different time periods *(*n.e.=observed but not estimated; \*Incubation Time=number of days from first observation of infected blood cells on stained blood smears to the peak of the disease).

Then, Lina developed the disease after an incubation period of 29 days and recovered within a month, exhibiting a slight decrease in PCV (less than 25%) on post-splenectomy day 90 due to a slight increase in parasitaemia by *A. ovis* (Fig. 4). The two sheep were transfused with blood from a healthy donor sheep and treated every two days for a week with oxytetracycline (Terramicina long acting 1000 mg) and dexamethasone (Desashock Fortdodge Animal Health S.p.a., 80mg single dose). Both Lina and Zoppina quickly recovered from the disease, reaching normal blood values within four weeks, but, one month after their recovery, they had a relapse which they coped with successfully.

different sheep breeds, described in section 5, parasite density was estimated on thin blood

Fifteen days after the splenectomy, the general situation worsened and the animals became anorexic, staggering with a severe anaemia and dehydration. At the same time the RBC, Hb% and PCV values dropped (Tab. 5), and a number of organisms started appearing in the blood films (Rosalba showed a carpet of *A. ovis*; Stella a great deal of *A. ovis*; Lisa and Lola a great deal of *A. ovis* and a few *Babesia* spp.; Claretta a great number of *Theileria* spp.). Rosalba and Stella died of severe anaemia respectively 24 hours and 4 days after the diagnosis despite specific drugs and whole blood trasfusions with blood drawn from a donor subject. Claretta, Lisa and Lola showed less violent initial symptoms, the anaemic crisis was less severe and following a therapy with anti-protozoal drugs associated with desametazone they gradually began to eat and became clinically and haematologically healthy in 15-20 days. Since Lina, Zoppina, and Gilda, were included in the above cited experimental design to investigate the tolerance to *A. ovis*, they were constantly monitored and parasitaemia was recorded every two days after splenectomy. The cases of Lina and Zoppina allowed comparison between a mixed infection by *T. ovis* and *A. ovis* and an almost single infection by *A. ovis*. Interestingly, the two sheep coped differently with the infections. Though both animals were positive for *A. ovis* and *T. ovis* after splenectomy, the maximum of parasitized erythrocytes (MPE) by *T. ovis* peaked to 17% in Zoppina, while in Lina *T. ovis* caused a latent infection. Conversely, MPE by *A. ovis* in

Year of splenectomy 1994 1994 1994 1994 1994 2009 2009 2010 Incubation Time (days)\* 19 21 25 32 42 29 35 21 Max Temperature (C°) 39.80 39.80 39.60 39.40 39.20 39.40 39.20 39.60 Min PCV (g/dl) 7 7 10 10 11 10 11 10 PCV reduction (%) 75 74 56 55 57 61 75 60 Hb reduction (%) 73 74 58 53 52 55 72 54 Max parasitemia *A.ovis*(%) >70 >60 n.e. n.e. n.e. 36 15 60 Max parasitemia *T.ovis* (%) n.e. n.e. n.e. n.e. n.e. 3 17 2 Table 5. Summary of clinical findings recorded in eight sheep splenectomised in different time periods *(*n.e.=observed but not estimated; \*Incubation Time=number of days from first observation of infected blood cells on stained blood smears to the peak of the disease).

Then, Lina developed the disease after an incubation period of 29 days and recovered within a month, exhibiting a slight decrease in PCV (less than 25%) on post-splenectomy day 90 due to a slight increase in parasitaemia by *A. ovis* (Fig. 4). The two sheep were transfused with blood from a healthy donor sheep and treated every two days for a week with oxytetracycline (Terramicina long acting 1000 mg) and dexamethasone (Desashock Fortdodge Animal Health S.p.a., 80mg single dose). Both Lina and Zoppina quickly recovered from the disease, reaching normal blood values within four weeks, but, one

month after their recovery, they had a relapse which they coped with successfully.

**Splenectomized sheep**  Rosalba Stella Lisa Lola Claretta Lina Zoppina Gilda

film and expressed as the percentage of parasitized red blood cells.

Zoppina was less than a half that of Lina (Tab. 5).

Fig. 4. Trend of PCV values and parasite densities (*An=Anaplasma ovis* and *Th=Theileria ovis*) expressed as percentage of red blood cells parasitized in Lina and Zoppina, the two splenectomized sheep monitored over a five week period after splenectomy.

During the recovery period, clinical examination revealed only pale mucous membranes. The pattern of evolution of the disease and recovery in Gilda was almost the same as in Lina except for a higher level of parasitaemia. In this instance *A. ovis* was the sole agent of the illness of our sheep. Secondly, the increase in *A. ovis* is apparently slowed down by the presence of *T. ovis* which seems to have a *'*buffer effect*'* on the appearance of anaplasmosis but not in tempering its severity. Three points emerge from these results: i) in contrast to babesiosis and to the literature of some years ago (Radostits et al., 2000), hemoglobinuria did not occur in any of the seven severe cases of anaplasmosis; ii) treatment produced an immediate reduction in parasitaemia without leading to a complete clearance of the parasites; iii) disease relapse might be accounted for by the persistence of *A. ovis.* 

Though not a novelty, in areas with enzootic erythrocytic parasitoses, even apparently healthy breeding animals may host pathogens and show tolerance and/or premunition to them without presenting with circulating parasites. This is a limit that should always be taken into account and may constitute a complication for any stress-associated situation. Anaemia secondary to anaplasmosis may evolve in a remarkably violent fashion probably due to the mechanism effected by the reticulo-endothelial system (RES) virtually with no

Tolerance to Tick-Borne Diseases in Sheep:

n.s., not significant.

major role also in the pathogenesis of TBD.

phenotype for disease resistance is difficult.

turnover of older and less efficient red blood cells.

Highlights of a Twenty-Year Experience in a Mediterranean Environment 463

anaemia (Pieragostini & Petazzi, 1999). So if it is a matter of fact that from the numerical point of view, the two breeds' responses to anaemization are to a large extent not very dissimilar, the greater efficiency of the local breeds is beyond doubt. It is not to be excluded that this may be identified in their greater capacity to cope with anoxemic stress, both by the production of red globules enriched with haemoglobin and maybe also by accelerating the

**Contrasts Haematological parameters** 

Within Altamurana n.s. n.s. n.s.

Within Romanov \*\*\* \*\*\* \*\*\*

Between Altamurana and Romanov \*\*\*\* \*\*\*\* \*\*\*\*

calculated by the regression analysis performed to compare the two breeds, Altamurana and

The reading of these haematological aspects should be looked at without losing sight of the general aspect of the overall comparison between the two breeds. From this point of view, the considerable difference in absolute values, apparently almost negligible as regards the curve trends, becomes very striking in the comparison between the general situations of overall well-being of the two breeds compared. The Altamurana sheep continued to exhibit apparent good health when subjected to anaemization, at ease with their surroundings, ready to feed and drink. Conversely, the Romanovs exhibited a serious dulling of the senses and lack of reaction once anaemization was achieved; this necessitated support treatments with rehydrating solutions to allow them to overcome their state of anergy (Pieragostini & Petazzi, 1999). These results strongly support the hypothesis that, beyond the environmental factors such as stress, nutrition and other conditions, which in general facilitate infections (Agyemang et al., 1990; Bennison et al., 1998; Oppliger et al., 1998) and which are supposed to be particularly relevant in the case of non-native breeds, genetic predisposition plays a

**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

Table 6. Statistical significance of the differences between the correlation coefficients

Romanov, with respect to the hematological parameters RBC (Red Blood Cells), Hb (Haemoglobin) and PCV (Packed Cell Volume). \*\*\*P<0.001; \*\*\*\*P<0.0001;

RBC Hb PCV

haemolysis. In our small experience the use of desametazone had a beneficial effect as it reduced the general response to the stimulation of the pathogen and particularly macrophage activity and improved red blood cell membrane response. Several years have elapsed since the first experiment and Lola, Lisa and Claretta got back to 'normal life' and, before their death, caused by old age, they showed no signs of disease which might have been related to haematological parasitosis. As to Lina, Gilda and Zoppina, they are back in the flock following a normal breeding and reproductive cycle. With no doubt the spleen naturally acts as an immunologically active filter-pad countering even severe red blood cell deprivation; its activity is particularly prominent in the presence of antibodies given that even after splenectomy these animals were still able to resist local diseases. There are grounds to believe that the animals may have a genetically derived tolerance to such instances based on active, diffuse and efficient structural systems which do not relate to one sole organ.
