HISTOPATHOLOGICAL, CLINICO-BIOCHEMICAL AND THERAPEUTIC STUDIES ON DIFFERENT TYPES OF MANGE IN DOMESTIC RABBITS

Assiut University web-site: www.aun.edu.eg

HISTOPATHOLOGICAL, CLINICO-BIOCHEMICAL AND THERAPEUTIC STUDIES

 ON DIFFERENT TYPES OF MANGE IN DOMESTIC RABBITS

RANIA I. MOHAMED ¹; DALIA IBRAHIM MOHAMED ²; MUSTAFA KHALIL ³;             NABILA M. EL MASRY ⁴ and NESMA RASHEED¹

¹ Department of Pathology, Animal Health Research Institute, Agriculture Research Center,

Egypt, Mansoura Provinicial Lab.

² Department of Biochemistry, Animal Health Research Institute, Agriculture Research Center,

Egypt, Zagazig Provinicial Lab.

³ Department of Poultry Diseases, Animal Health Research Institute, Agriculture Research Center,

 Egypt, Mansoura Provinicial Lab.

⁴ Department of Parasitology, Animal Health Research Institute, Agriculture Research Center,

Egypt, Mansoura Provinicial Lab.

Received: 31December 2016;       Accepted: 19January 2017

INTRODUCTION

Sarcoptic mange, belongs to the family sarcoptidae is infectious disease which can threaten human  and  animal  health  globally  (Alasaad  et al.,

Corresponding author: Dr. RANIA I. MOHAMED

E-mail address: dr_raniaibrahem@yahoo.com

Present address: Department of Pathology, Animal Health Research Institute, Agriculture Research Center, Egypt, Mansoura Provinicial Lab

2011). In Egypt, mange (Sarcoptes and Psoroptes) in rabbits considered to be second to coccidiosis importance with high losses. (Ezzat, 1955). This dermatological Problems is occurred due to hot and humid climates (Aulakh et al. 2003). Moreover, overcrowded living conditions and Poor hygiene are significant factors for infestation by Sarcoptes mites (Mc Carthy et al., 2004). Mites generally spread from rabbit to rabbit by direct skin contact between infested and non infected rabbits or, through contact with the environment (Panigrahi and Gupta, 2013). Severe infestation especially in young or debilitated animals cause high mortality (Bornstein and Samuel, 2001). It characterized clinically by Pruritis, alopecia and prolonged illness. The animal become emaciated and may even die due to Cachexia. (Roy et al., 2001). Sarcoptes Scabiei is a deep burrowing mite in epidermis causing intense itching, purities, pyodermatitis, crust formation, Scar production, thickening of skin of affected areas, it causes infestation which affects ears, nose and feet beside area around genitalia, (Kachhawa et al., 2013). Psoroptes cuniculi, causes infestation primarily in the ears(Kyung-Yeon and Oh-Deog, 2010) which may cause extensive pyodermatitis in the ear or on the body surface of rabbit (Siegfried et al., 2004). It lives in the external auricular meatus where it is may fed on the serous exudate, skin secretion, and blood (Fichi et al., 2007 and Kyung-Yeon and Oh-Deog, 2010). Psoroptes mange is characterized by hypersensitivity reaction by the host to mite antigenic material causing inflammation, surface exudation, severe puritis, anorexia emaciation, fur loss, restlessness, biting and scratching of affected areas.

In severe cases, it causes meningitis and may be fatal when complicated by secondary bacterial pathogens (Kyung –Yeon and Oh-Deog, 2010 and Shang et al., 2014).

Two forms of P.cuniculi infestation are recognized in rabbits: i) typical Psoroptic (ear canker) which is confined to the ear canal and the pinnae ii) extra –auricular mange, spreading over the body of the rabbits (Bates, 1999). Infestation may be chronic or even subclinical. In recent years attention has been focused on the use of Propolis as health supplement (Bankova, 2005).

Several strategies have been directed for treatment of mange caused by P. cuniculi, ranging from the use of acaricides, and essential oils, to vaccines (George et al., 2014). However, therapy and control of both human scabies and animal mange are still based mainly on the use of drugs and macrocyclic lactones such as ivermectin, selamectin, moxidectin, and eprinomectin(Wen et al., 2010 and Borges et al., 2013). The use of drugs, such as ivermectin has disadvantages, since it has both, genotoxic and cytotoxic effects. In addition, their widespread use induces resistance, accompanied by environmental pollution (Coles and Dryden, 2014).Such side effects of chemical acaricides have prompted a search and research efforts to discover new alternatives and effective compounds (Khater et al., 2013).

It is known that when reactive oxygen species (ROS) generation over-leads the antioxidant defense, the free radicals can then interact with endogenous macromolecules and alter the cellular functions (Muthukumaran et al., 2008). Free radicals and oxidative stress are involved in the pathogenesis of various diseases (Sies and Krinsky, 1995) including sarcoptic mange and Psoroptes mite (Gurgoze et al., 2003). However to our knowledge, whether P. cuniculi infestation causes oxidative stress or not has not been reported.

Propolis (bee glue) is a resinous hive product collected by honey bees from exudates and buds of plants and mixed with wax and bee enzymes (Burdock, 1998). Propolis has various biological and pharmacological properties, which had been associated with the presence of flavonoids and aromatic acids and esters (Salomao et al., 2004). Propolis possessed variable biological activities as antimicrobial (Abd El Hady and Hegazi, 2002), anti-inflammatory (Park and Kahng, 1999), antioxidant (Kanbur et al., 2008), antiparasitic (Hegazi et al., 2007) antibacterial (Hegazi and Abd El Hady, 2002); fungicidal (Hegazi et al., 2000); antiulcer and anti-tumor (Hegazi et al., 1998).

Therefore, the present work was conducted to evaluate the therapeutic effect of 10 % propolis ointment (as a local immunostimulant) against naturally infested rabbits with both body mange (Sarcoptes scabiei) and ear mange (Psoroptes cuniculi) in comparison with subcutaneously injected ivermectin. Hematological, biochemical, and histopathological investigations were done to check the propolis effect.

MATERIALS AND METHODS

Animal Used.

Forty male Commercial rabbits of different breeds (New Zealand and Baladi), their weights of 1.5–2 kg and 3-8 months old were used in this experiment. All rabbits were fed on balanced rations and a clean source of water ad libitum. The sampling procedures adhered to institutional ethical and animal care guidelines, and all methods were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, according to Nong et al. (2013). Ten of these animals were healthy, allocated to the control group and the remaining 30 were naturally infested by Psoroptes cuniculi and Sarcoptic scabiei were obtained from some farm affected by an outbreak. The severity of the infection was determined according to clinical and laboratory examination and the scoring system described by Ulutas et al. (2005).

Topical use of Propolis.

10 % propolis ointment prepared by adding 10 gm propolis to 90 gm soft paraffin then used as topic for 3 successive days according toAbd El Aal (2005).

Experimental design

Rabbits were classified into four groups (ten rabbits each). The first group (group 1) was designated as the healthy or negative control group (non-infested by sarcoptic scabiei or psoroptic cuniculi mites and non-treated). Each rabbit in groups 2, 3, and 4 was naturally infested with Psoroptes cuniculi and Sarcoptic scabiei. The second group (group 2) was not treated and taken into account as the positive control group. The third group (group 3) was subcutaneously injected with ivermectin (IVR) 1 % (Ivomec®, Merk Sharp and Dohme Agvet Inc.) two doses one week in between, at dose 400 µg/kg according to Galdhar et al. (2015). Finally, group 4 was treated topically with 10 % propolis ointment for three successive days, according to Abd El Aal (2005).

Erythrogram and Biochemical Analysis.

Two blood samples were obtained from the ear vein of each rabbit after 10 days post treatments. The first sample was anticoagulated and used for the determination of the erythrocytes count, packed cell volume, hemoglobin concentration, and leucocytes by using Coulter (MEDONIC CA620) according to Feldman et al. (2000). The second sample was collected for serum separation and determination of serum biochemical constituents. Aspartate aminotransferase (AST) and Alanine Aminotransferase (ALT) were determined on semi-auto-analyzer (Microlab-300, Merck) using commercial diagnostic kits manufactured by Merck (Reitman and frankel, 1957). Plasma malondialdehyde (MDA) levels were determined as described by Yoshioka et al. (1979); glutathione transferase (GST) was determined as described by Paglie and Valentie (1967) and superoxide dismutase SOD was assayed as described by Sun et al. (1988).

Parasitological examination

Skin scraping from body lesions and ear waxes were examined directly by microscope searching for moving living mange. The identification of the causative mite was carried out by preparation of permanent specimens using 10% sodium hydroxide in water bath for clearing from debris for fixation. The organisms were identified according to the keys of Baker et al. (1956) and Soulsby (1982).

Clinical and pathological examinations

Observations and recording of clinical signs, weight, and general conditions of control and infested and treated rabbits were done. Specimens from skin and ears of normal, clinically infested and treated rabbits( were collected after 10 days post treatment) and fixed in 10% neutral buffered formalin solution, dehydrated in gradual ethanol (70-100%), cleared in xylene and embedded in paraffin. Five micron sections were prepared and then routinely stained with hematoxlyin and eosin (H&E) according to Suvarna et al. (2013), then examined microscopically.

Statistical analysis

Data were analyzed using the one way variance of analysis (ANOVA) test and Duncan test according to Snedecor and Cochran (1982).

RESULTS

Parasitological results:

Result in table (1) revealed that out of 200 examined rabbits, 30 (15%) were infected with mange. The incidence of body mange was 4% and ear mange was 5%, while the incidence of mixed infestation was 6% (Table2). Rabbits infested with both ear and body mange showed emaciation and scratch of lesions due to intense pruritis, which lead to presence of scabs or dried crusts.

Infestation with the mange Sarcoptic scabiei (Fig.1), Psoroptic cuniculi (Fig.2) from infested rabbits were diagnosed by skin scrapping and microscopically, the results were tabulated in Table3. The seasonal dynamics of mange among rabbits in winter and spring was 10% and 18.5% respectively. However, microscopically examination of skin scraping collected from treated parts after 10 days post treatment were negative for adult mites and its developmental stages.

Table 1: Prevalence of mange amonge rabbits.

This variation of incidence might be attributed to different management in the rabbit farms as the mange is a disease of poor condition animals, and due to variation of strain resistance of rabbit to mange infestation.

Table 2: Incidence of ear, body and mixed infestation with mange amange rabbits.

 Table 3: Seasonal dynamics of mange amonge rabbits in two seasons.

The prevalence of rabbit mange also varied seasonally in our study this variation might be attributed to the favorable temperature and humidity in spring.

Hematological Results:

Rabbits infested with Sarcoptes and Psoroptes showed a significant decrease in RBCs count and PCV %values, while WBCs count exhibited significant increase compared to control group. Along the period of treatment, no significant changes were demonstrated in the values of Hb content (Table 4). Treatment with ivermectin and topically application of propolis recover RBCs count, PCV and WBCs count to normal level after 10 days post treatments.

Table 4: Erythrogram in different group of rabbits infested with mange and treated with either ivermectin or propolis (Mean ± SE,n = 5).

Values are means ± standard error (SEM) for 5 different rabbits per each treatment. Values are statistically significant at *p<0.05 Vs. control; #p<0.05 Vs. diseased.

Antioxidant results

Rabbits infected with Psoroptes cuniculi and Sarcoptic scabiei showed a decrease in antioxidant enzyme levels SOD and GST but exhibited an increase in MDA compared to normal rabbits as shown in Table (5). Treatment with both ivermectin and propolis restored these parameters to normal level after 10 days post treatment.

Table 5: SOD, GST and MDA levels in plasma of rabbits infested with mange and treated with either ivermectin or propolis (Mean ± SE,n = 5).

Values are means ± standard error (SEM) for 5 different rabbits per each treatment. Values are statistically significant at *p<0.05 Vs. control; #p<0.05 Vs. diseased.

Biochemical Reults

Table 6. presented significant increase in liver enzymes AST and ALT in rabbits infected with Psoroptes cuniculi and Sarcoptic scabiei however treatment with either ivermectin or propolis resulted in restoration in biochemical parameters to normal level.

Table 6: ALT and AST levels in serum of rabbits infested with mange and treated with either ivermectin or propolis (Mean ± SE, n= 5).

Values are means ± standard error (SEM) for 5 different rabbits per each treatment. Values are statistically significant at *p<0.05 Vs. control; #p<0.05 Vs. diseased.

Clinical and Pathological results:

1. Body mange (Sarcoptes scabiei) infestation:

A- Clinical signs and necropsy findings:

Multifocal areas of alopecia areas containing scales were seen scattered on legs, face, lips, nose, ears, eyelid and fur (fig. 3-6). The infected rabbits were emaciated and showing scratching of the lesions due to intense pruritis. Greyish or whitish exudate, scabs or dried crusts sometimes could be seen. The morbidity and mortality rate among infected breeding flocks ranged from 5-8 % and 0.5-2% respectively. Necropsy showed scales, crust formation, scar production, thickening of skin of affected areas with tunnels formation.

B- Microscopically:

Lesions characterized by hyperkeratosis, acanthosis and subepidermal dermatitis. Excess keratin production, with necrotic debris and sloughed epithelia mixed with developmental stages of the parasite could be seen on the body surface and within epidermal burrows (Fig.7 & 8). Hyperplastic epidermal cells with formation of keratin cyst could be seen. Sometimes bacterial colonies could be seen in exudate and epidermal tissues. Intense dermatitis manifested by leukocytic infiltrations mainly lymphocytes, macrophages, together with hyperplastic sebaceous glands and dilated blood vessels were noticed (Fig, 9 & 10). A few heterophilic infiltrations and hemorrhages could be seen accompanied by exfoliation of the epidermis.

After 10 days post treatment:

A clinical and macroscopic findings of treated rabbits showed improvement in skin lesions begins from 3rd days post treatment with either ivermectin or propolis manifested by reduction or absence of scales, mite stages (adult nymphs, larvae and eggs) and beginning of healing which usually nearly completed after 10 days.

Microscopic picture, (10 days post treatment) by ivermectin or propolis:

Pathological findings in treatment by ivermectin or propolis showing similar findings represented by regenerated skin.

Thin epidermal celayers were regenerated together with edematous dermal fibrous tissue containing little inflammatory cells mainly lymphocytes. In addition skin adnexa usually seen replaced the majority of the skin lesions (Fig,11 & 12) were observed in both ivermectin or propolis treatment.

2. Ear mange infestation (Psoroptes cuniculi):

A- Clinical signs and gross lesions:

The disease was seen as sporadic cases among infected breeding and laboratory flocks, the infected rabbits revealed pruritus with scratching of the ears and head. Grayish, dry foul smelling exudate usually seen inside the ears. Other rabbits had thickened crusts which obstruct ear canal. The adjacent skin usually inflamed, ulcerated or scaly.

B-Microscopically:

Parasitic elements embedded within exudate from necrotic debris and inflammatory cells or free on the ulcerative surface and surrounded by intense inflammatory cells infiltration in subepithelial tissues (Fig, 13). The adjacent epidermal cells were hyperplastic with parakeratotic and acanthotic changes. Sometimes, parasites penetrate deeper in skin and underlying structures of the ear. Subepidermal tissues were edematous and highly infiltrated with lymphocytes and heterophils together with extravasated erythrocytes (Fig, 14).

After 10 days post treatment:

Nearly the same results were observed when using propolis and ivermectin. Gross mites reduction and complete absence of exudate from ear canal and mite stages.

Microscopic picture (10 days post treatment) by Ivermectin or propolis:

The majority of skin lesions showed mature fibrous tissues with islands from inflammatory cell aggregations and dilated blood vessels beside skin adnexa covered by several layers from non keratized epidermal cell layers, these pathological findings showed similar findings in treatment either by ivermectin or propolis as described in (Fig. 15&16).

DISCUSSION

Mange in rabbits due to Sarcoptes Scabiei or Psoroptes cuniculli considered a worldwide obligatory contagious ectoparasites (Perruci et al., 2005),which parasites on the surface of epidermis of rabbits causes pruritis and dermatitis, so it is classified as a major skin disease infest both young as well as adult rabbits (Siegmu, 1979).

Parasitological examination and skin scrapping revealed mange prevalence as 10% in winter while it was 18.3% in spring. These results were in agreement with Zelek and Bekele, (2001),and Smith, (2009), who mentioned that symptoms of mange were common during cold months. Regarding clinical signs and necropsy findings in infested rabbits showed characteristic skin lesions in most of the body parts accompanied emaciation and scratch lesions due to pruritis. These mites produce their pathologic effect by burrowing activity and mechanical damage during the excavation, Irritant action of their secretions and excretions while allergic reactions was resulted from extracellular products especially release of interleukin -1 (Wall and Shearer, 1997).

Crusted lesions contained large number of the parasite below the crusts are due to weak hypersensitivity reaction (Henry, 1996).

It is known that inflammatory cells are increased as a result of inflammation in rabbits with mange; recruited neutrophils and macrophages produce reactive oxidants such as hydrogen peroxide (H2O2), hypochlorite, and oxygen radicals. These reactive oxygen substances produced by cells of immune system showed potent cytotoxic effects on parasites and other pathogenic organisms (Gurgoze et al., 2003). Lipid peroxidation is well-established mechanism of cellular injury is used as an indicator of oxidative stress in cells and tissues. Lipid hydroperoxides are by-products of lipid peroxidation and increased levels of lipid peroxidation products are associated with variety of diseases such as parasitic infestations (Kiral et al., 2005). Lipid peroxidation can be harmful for skin due to alterations in the structure and permeability (Portugal et al., 2008).

The elevation in MDA levels in Psoroptes and Sarcoptic scabiei infested rabbits, the lipid peroxidemediated skin lesions. The decreased level of the body antioxidant (GSH) and reduced activities of the antioxidant enzymes for instance GST, CAT, and SOD imply that Psoroptes mite-infested rabbits are in a state of significant oxidative stress, and an altered antioxidant defense mechanism is under operation. States of oxidative stress have been demonstrated with various parasitic skin infestations including Sarcoptic mange (Saleh et al., 2011) and Psoroptic mange (Dimri et al., 2010).

Amelioration of the altered oxidant/antioxidant balance towards normal in ivermectin and propolis treated rabbits indicated potential antioxidant action of propolis. In the present study, we demonstrated both faster clinical and parasitological recovery in the ivermectin and propolis treated rabbits. Presented microscopical examination of skin scraping collected from treated parts after 10 days of treatment were negative for adult mites and its developmental stages and this in agreement with Amer and Abd El Aal (2005)who studied the effect of propolis in rabbits mange and found that use of 10 % propolis ointment for 3 successive days resulted in complete reduction of clinical signs. Moreover, Garedew (2003) attributed these results to the acaricidal effect of propolis on body or ear mite, due to remarkable reduction in metabolic rates of mites. Presented results coincided with Garedew (2003) who stated that lethal effect of honey bees increased with increasing the concentration and the contact time achieving 100% mortality at 10% propolis concentration. Antioxidant vitamins in propolis generally enhance various aspects of cellular and non-cellular immunity. β-carotene has significant antioxidant activity and can effectively quench the free radicals (Sharma and Sharma, 2009). As a hydrophobic antioxidant that incorporates into lipid environments, vitamin E significantly decreases lipid peroxidation in different organ and body fluid (Kara et al., 2008).Potential antioxidants include either natural free radical scavenging antioxidant enzymes or the agents which are capable of augmenting the activities of these enzymes, including the GSH-Px, GST, SOD, and CAT. In the current study, propolis treatment revealed potential enhancements in the antioxidant defense systems. The restoration of antioxidants and amelioration of lipid peroxidation in propolis treated rabbits might be involved in the faster clinical and parasitological cures.

Infested rabbits had higher MDA levels and lower GST and SOD levels than normal rabbits. This result indicated that P. cuniculi infestation caused oxidative stress. The increase in MDA levels was most probably due to the increase in the biosynthesis of the enzyme caused by H2O2 and other free radicals in P. cuniculi infestations; this is in agreement withKanbur et al. (2008) who reported that Infestation stimulated oxidative stress. In the infested rabbits exposed to ivermectin and propolis, plasma MDA values decreased and GST and SOD levels increased, in comparison to values pertaining to non-treated rabbits. This phenomenon might be explained by the healing of infected rabbits following treatment with ivermectin and propolis. Singh et al. (2012) concluded that significant alteration of oxidant/antioxidant balance is a factor in the pathogenesis of P. cuniculi infestation of rabbits, and recovery can be enhanced by combining ivermectin treatment with propolis supplementation.

Regarding serum enzyme activities, AST and ALT showed significant gradual increase in infested rabbits along the period of experiment in comparison with control group. This result agreed with Talas and Gulhan (2009). The demonstrated result in the present study revealed that administration of propolis had no toxic effect on rabbit. A restoration in serum ALT and AST level was observed in propolis-treated groups; similar result was observed by Eraslanet al. (2007). Results of liver enzymes activities support that propolis able to induce hepatoprotective effects which are similar to those of the previous work on propolis that act as hepatoprotective against d-galactosamine, and paracetamol induced liver damage in rats and mice, respectively (Sugimotoet al.,1999).

The microscopic findings of body mange characterized by hyperkeratosis, acanthosis and subepidermal dermatitis, excess Keratin formation due to Sarcoptic mange results were in accordance with those obtained by Chandey et al. (2000) and Oraon et al. (2000).

Treated rabbits with ivermectin (400ug/k) showed improvement in skin lesions begins from the 3rd day post treatment leading to reduction or absence of scales and mite stages, this attributed to binding at ivermectin to glutamate gate and gamma-ammino butaric acid (GABA) gates chloride channels in the nervous system of mites resulting in hyper polarization of cells, Paralysis and finally death of mites. There results were in agreement with those obtained by Aulakh et al. (2003).

Rabbits infested with ear mange showed inflammation of skin, thekened crusts formation (Pruritic dermatitis), foul exudates inside the ear even obstruct the ear canal and loss at weight.

Psoroptes mites can complete their life cycle on the epidermis and then bore it, and feed on skin food and fluid. Skin fat resulted in release of toxins which lead to chronic allergy leading major injuries, scratching, reduce animal appetite and cause secondary infection (Joel and William, 1983).

Microscopic findings of ear mange showed hyperplasia, parakeratosis and acanthosis of the adjscent skin to ear, these results were similar to those obtained by Lossen et al. (1999).

Dermatitis of the thickened external ear is due to inflammatory cells infiltration, even ulcers and necrotic debris was created by three mechanisms:i) Part of the lesions is due to direct mechanical stimulation of the parasites and mucus from mange.ii) The allergic reaction that is created by immediate or delayed sensitivity in rabbits.iii). The damage that infested rabbits provides due to excessive itching and rubbing to objects.

After treatment with ivermectin, gross mites reduction occurred, and complete absence of exudate from ear canal. These findings were in agreement with Acar et al. (2007), as they successfully treated clinical signs manifested by rabbit with ivermectin.

It could be concluded that the efficiency of propolis ointment in treatment of Psoroptes cuniculi mite (ear mite) Sarcoptic scabiei which showed sluggish movement, stopped then lethal effect, Moreover, on the third day of treatment showed complete reduction of clinical lesions Garedew (2003) attributed these results to the acaricidal effect of propolis on body or ear mites, due to remarkable reduction in metabolic rates of mites. Presented results coincided with Garedew (2003) who stated that lethal effect of honey bees increased with increasing the concentration and the contact time achieving 100% mortality at 10% propolis ointment. Amer and Abdel Aaal(2005) augmented the present results that application of propolis ointment at 10 % concentration resulting in complete reduction of clinical signs from the third day of treatment. They inferred that, the propolis extract succeeded to treat rabbit mange, so it was considered a good acaricidal.

CONCLUSION

We concluded that treatment of both types of mange in rabbits either by ivermectin (400 µg/kg) or 10 % propolis ointment causes improvement of clinical signs, pathological lesions and biochemical parameters in infested rabbits. Moreover, microscopical examination of skin scraping collected from treated parts after 10 days of treatment were negative for adult mites and its developmental stages Thus, propolis can be used as an efficient and safe natural remedy for controlling mange in rabbits with avoidance the problems of chemical drugs.

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