The species of the family Leporidae (rabbits and hares), from the order Lagomorpha, include both individuals that have been domesticated as companion and/or production animals used in the food or fur industry as well as in biomedical research [1
], and those wild animals that are part of natural ecosystems. Diseases have been closely linked to lagomorphs. In the raising of rabbits in rural areas and in wildlife lagomorphs, disease has always been present [2
] (pp. 481–498). However, the social and economic changes of the past few decades have caused lagomorph medicine to evolve significantly and to have become more important than ever, due to increased productivity and economic needs [3
] of the farm industry.
The health of industrially farmed rabbit populations is of great relevance from a productive and financial point of view [4
]. Many animals are culled from farms as a result of disease or low productivity. Despite the significant progress experienced in the field of genetics, feeding and management, the increase in productivity, the high selection of breeds and their crosses make the onset of diseases unavoidable [5
]. This implies an additional expense in a sector where, today, production costs outweigh benefits. In addition, the increasing demanded for ecological cuniculture, where the use of synthetic drugs is not allowed, makes the knowledge of both the associated different disorders and proper health management key factors in this sector. In the case of wildlife lagomorphs, their importance lies in their ecological value as species within the food chain, their economic value for hunting, and their being a reservoir of zoonoses and economically important diseases [7
]. In this sense, the evaluation of dead animals within epidemiological surveillance plans is crucial for the evaluation of the health status of wild populations. At the same time, the extraordinary boom that pet rabbits have had recently in European and North American societies emphasizes the importance of knowledge of the different associated diseases from a clinical and public health (zoonosis) point of view.
In most cases, in both domestic and wild species, the study of the disease is approached from the perspective of the community (without belittling valuable individuals, such of pet) and integrated into the medicine of production and/or conservation. This makes the mortality risk factors, causes of death and prevalence rates of the different pathological conditions variables of interest from a health and welfare perspective [9
] (pp. 237–245). The greater our knowledge is, the better we can diagnose rabbit and hare diseases, enhance their prevention, and choose the appropriate control method for a specific population [10
Despite the accumulation of information on lagomorph medicine and pathology, comprehensive published compilations on lagomorph diagnostic cases are relatively scarce. The purpose of this study is to report a retrospective evaluation of diagnostic cases found in lagomorphs (rabbits and hares) received over an 18-year period at the Department of Animal Health of the Faculty of Veterinary Sciences of the University of León, a region in the Northwest of Spain
Where there is a significant lagomorph population, both in wildlife and farms. Our aims were both to determine the prevalence of the diagnosed diseases considering risk factors such as species, age, sex, time of year and origin, and to characterize those diseases or illness through their gross and histopathological features after necropsy. Our challenge was to contribute to the knowledge and prevention framework of lagomorph disease.
2. Materials and Methods
From 19 January 2000 to 27 November 2018, we obtained the data of our study from a total of 325 necropsies performed on diseased and dead lagomorphs referred to the Diagnostic Service of the Department of Animal Health of the Faculty of Veterinary Sciences (University of León, Spain). Whenever possible, the referring owners or veterinarians of each animal submitted to the laboratory provided a brief clinical history (duration and type of clinical signs, number of animals affected, etc.), the sex, age and exact date of death of the animal. Based on this information, the animals were classified according to the species (hares or rabbits), sex, age category (young; less than 7 months of age, adults; over 8 months) [11
] (pp. 189–249), origin (farm; meat production animals raised in a traditional way and/or by industrial breeding farms; wild and pets) and time of year in which the reception of the animal was registered (spring–summer: 20 March–20 September; autumn–winter: 21 September–19 March). The vast majority of farm and pet rabbits belonged to the New Zealand breed. The distribution of animals according to the categories described is shown in Table 1
. All data were recorded before performing the regulated necropsy of each animal included in the study.
Animal Care and Use Committee approval was not obtained for this study because data were gathered from animals raised under commercial conditions and wild animals were recovered by the Environment Agents of the Junta de Castilla y León, regional government, fulfilling European, Spanish, and regional recommendations and laws on animal welfare and wildlife management.
2.2. Necropsy, Histopathological and Microbiological Diagnostic Workup
The 325 animals included in the study were submitted to the Diagnostic Laboratory mostly within 24 h of death. Many of the specimens that arrived at the laboratory from commercial rabbit farms showed different degrees of autolysis because dead rabbits were removed daily from the farm facilities to the area for rendering (refrigerator and freezer) as a biosecurity measure [12
] (pp. 1–11), [13
] (pp. 337–369). In this study, only carcasses in good condition were included to accurately describe the gross and histopathological findings after necropsy and identify the exact cause of death or illness. The necropsy protocols used were those described by Corpa (2009) [14
]. Gross examination was performed on all animals and organs following a standard protocol. A systematic and complete sampling of tissues from each animal was carried out for histological analysis. All samples were fixed in 10% neutral buffered formalin for at least 48–72 h and stored until histological processing. In turn, when suspected gross lesions were observed, tissue samples were collected in sterile tubes and stored at −20 °C until microbiological analysis took place.
For histopathological examination, tissue samples were briefly washed with 10% phosphate buffered saline (PBS) solution, dehydrated in alcohol and embedded in paraffin wax (EI LEICA TP1020 Automatic Tissue Processor®, Barcelona, Spain). Serial 5 μm sections from all specimens were mounted on glass slides (Super-Frost, Menzel-Gläser, Braunschweig, Germany), stained with hematoxylin and eosin (HE) and examined by light microscopy. Additional stains were performed as needed and most commonly included Gram, periodic acid-Schiff, Giemsa and Ziehl-Neelsen stains. Lesions were examined by the authors, who included three fully qualified European College of Veterinary Pathologists (ECVP) diplomates (V.P, J.F.G.M and N.C).
For microbiological studies, after flame the tissue samples, one gram of each tissue and/or pathological material was placed in a sterile airtight bag with 3 mL of sterile 10% PBS and homogenized for 5 min (Stomacher 80 Biomaster®, Lardero, Spain). Samples were cultured on Blood and MacConkey agar plates (supplemented with streptomycin when necessary; bioMérieux, Madrid, Spain) and incubated aerobically and/or anaerobically at 37 °C for 24–48 h. Direct observation of the bacterial colonies was carried out after specific staining (Gram). Bacterial strains were identified by analyzing the macro and micromorphological characteristics, physiological, enzymatic and/or biochemical tests of the different microorganisms with the help of API gallery systems (Biomerieux®)and, according to the type of bacterial agent, with visual reading and analysis in API software Web version 4.0 (Biomerieux®) or automated biochemical tests by Vitek® System Cards (BioMérieux®). Between 2014 and 2018 all isolates were analyzed in a Bruker DaltonikUltrafleXtreme MALDI TOF/TOF (desorption/ionization time-of-flight mass spectrometry) system, which obtained one spectrum per sample used to assess the suitability of this spectrometric approach for identification at the species level. Each spectrum was acquired using FlexControl software (Version 3.4) in automatic mode in a random sampling pattern. The identification of all the clinical isolates in this study was performed by MALDI Biotyper Real Time Classification software. The reliability of the identification was evaluated from the log (score) values, calculated with the MALDI Biotyper software mentioned above.
Diagnosis of viral, parasitic and toxic diseases was based on the observed clinical-epidemiological and histopathological features. Identification of parasitic agents was carried out according to their morphological characteristics.
2.3. Statistical Analyses
The different processes diagnosed were expressed as percentages with respect to the total number of conditions detected in each group. Non-parametric statistical methods were used to compare between groups. Specifically, the Pearson Chi-squared with Yates continuity correction test was used to contrast the relationship between the detection frequency of each condition with the different variables considered (species, age category, sex, time of year and origin). Where necessary, in a second step, Pearson Chi-squared with Bonferroni correction post-hoc comparison was performed to determine the differences among the groups analyzed [15
] (pp. 243–355). p
-values < 0.05 were considered to be statistically significant.
All statistical analyses were performed with the R software version 3.5.3 [16
The type, frequency and pathological features of the conditions identified in this work were in agreement with the most important lagomorph diseases already reported in other European countries as well as in other referenced studies [10
]. In our case, the data must be interpreted in the light of their retrospective nature. Studied animals were submitted to the laboratory randomly over time, so there is a bias in terms of origin or species, the most commonly examined animals being farm rabbits.
Parasitic diseases were the conditions most frequently detected, particularly those causing digestive disorders. Clinical disease was observed mostly in young animals affected by intestinal and hepatic coccidiosis or encephalitozoonosis, coming from traditionally managed rabbit farms. Factors such as poor hygienic sanitary conditions, inadequate management systems and poor health prophylactic protocols still present in this type of farms, together with immature immune systems in young animals, probably promoted those disease outbreaks [19
]. In addition, there was a marked seasonal pattern (mainly during the autumn–winter period), with favorable environmental conditions for the resistance and transmission of parasitic agents [21
] (pp. 343–356). Encephalitozoonosis was the most frequent parasitic disorder, and the second most common condition, of the study. The relevance of this disease acquires importance not only froma productive or animal welfare point of view, but also from its zoonotic potential [22
]. Most of the affected animals showed the subclinical disease and only a small number were reported to show the evident clinical symptoms that are usually associated with large parasitic loads [23
]. All the rabbits in which this disease was diagnosed were euthanized and no direct deaths were recorded. Altogether, these findings would suggest that these animals were well adapted to this microsporidian agent [23
]. A significant number of rabbits in which encephalitozoonosis was diagnosed were simultaneously affected by other diseases such as myxomatosis, pneumonic pasteurellosis or skin abscesses, or were highly parasitized. As described in humans, it seems feasible that immunosuppressive conditions might have encouraged infection and the clinical picture [24
]. In the cases of intestinal coccidiosis, the severity of the clinical and pathological findings made highly pathogenic species such as E. magna
and E. intestinalis
the most likely etiology. A significant number of animals with intestinal coccidiosis showed concomitant infections with E.coli
spp. The so-called “rabbit enteritis complex” was the fourth most frequently diagnosed condition and the second disease responsible for digestive disorders. Imbalances in intestinal physiology associated with nutritional disorders or by primary infections caused by some of these agents are responsible for these concomitant infections [26
]. In contrast, hepatoperitoneal cysticercosis, parasitic gastritis or mite infestations were significantly related to the adult age class and always seen in wild animals. The mild clinical signs found suggest a tendency towards a host–parasite equilibrium. The absence of hepatoperitoneal cysticercosis in farm animals found in this study has been associated with the needs of the parasite’s evolutionary cycle [27
spp. parasitosis is also influenced by host sex, since males were the most affected individuals, similarly to other wild populations [28
], without a clear explanation of this bias. High parasitic loads are associated with concomitant infections such as myxomatosis or RHD, which in this case were not observed. Although not investigated in the present study, the number of parturitions in female rabbits or the length of breast feeding, directly related to the individual physiological state, have been seen to influence the prevalence of psoroptic mange on farms [29
Bacterial diseases showed a presentation pattern similar to parasitic conditions, except that they were more prevalent among breeding females from industrialized farms. Bacterial mastitis, skin abscesses, pneumonic pasteurellosis and septicemic processes showed similar epidemiological characteristics. Bacterial mastitis was the most frequent female reproductive system disease in our study, followed by neoplastic processes and uterine rupture and torsions. Its incidence increases with age and the number of births [30
] (pp. 183–193). Similar etiologic agents were identified in these four conditions, all of them considered to be opportunistic agents that need other factors such as previous trauma to the nipples or skin, climatic stress (with a marked seasonal autumn–winter pattern), or overcrowding in the case of intensive breeding to trigger the onset of all these diseases [13
] (pp. 337–369). Skin abscesses were the most frequent dermal condition in farm rabbits, mainly associated with Staphylococcus
spp. and directly related to the presentation of generalized pyogenic septicemic processes [33
]. On the other hand, pseudotuberculosis and tularemia are rare diseases in farm animals while frequently diagnosed in wild animals, as in this study. The fact that examined cases appeared in subclinically infected adult animals could indicate some resistance to this disease and the need of concomitant factors for the occurrence of epizootic outbreaks of high mortality [34
]. Although in this study both diseases appeared more frequently in females, no references were found regarding sex predilection. Tularemia was observed only in wild hares, with a greater majority of cases in the spring–summer period, probably linked to favorable weather conditions for maintenance of the transmission vectors of this infection, as observed by the high tick infestations the affected animals presented [35
]. The absence of diagnosis of tularemia in wild rabbits from the same areas, where they share the same environment with affected hares, could indicate some resistance in this species. Taking into account that the clinical and pathological findings are very close to other diseases such as Tyzzer’s disease, listeriosis and pseudotuberculosis, there is a need for diagnostic confirmation by microbiological culture. The importance of this disease also lies in its potential zoonotic role [36
Epizootic rabbit enteropathy was the most frequently diagnosed condition of the study. The disease occurred as epizootic outbreaks within farms with a subacute presentation. Although this condition can occur in animals of any age, in our case, it was detected mainly in young individuals. In accordance with our results, it has been reported that the season of the year is directly related to this syndrome, with a greater number of cases in autumn–winter period due to increases in food intake [37
]. The pathogenesis of this disease is still unclear, although it is thought that it may have an infectious origin since the outbreaks are controlled with antibiotics. However, the main hypothesis is that any factor that favors intestinal dysbiosis may be behind the problem [38
]. Animals with this disease also presented pneumonic pasteurellosis, as a reflection of the opportunistic nature of this condition, which can exacerbate the digestive disorders.
Hepatic steatosis observed in young non-obese animals was linked to prolonged fasting due to poor management or stressors. However, the presence of this condition in pregnant obese rabbits with pregnancy toxemia was associated with a negative energy balance in the final phase of pregnancy, and where a certain seasonal trend has been seen, mostly in the winter–autumn period [39
]. The dystrophic calcifications observed in kidney and elastic arteries are associated with vitamin supplements rich in vitamin D, calcium and phosphorus and where age is usually a risk factor [41
]. Although mineralization in arteries is common, cartilaginous and bone metaplasia with bone marrow formation is rare and is usually associated with previous injuries (e.g., atherosclerosis), which in this case were not detected [42
RHD was the third most common condition detected in this study and, together with myxomatosis, the only viral disease. Currently, both disorders are thought to affect mainly wild species since the establishment of effective vaccination programs has greatly reduced their presence in industrialized farms. The cases of RHD remitted showed a marked seasonal pattern since they were more numerous both in spring and autumn, coinciding with the epizootic outbreaks of the disease in the wild, and with young and female animals as the most susceptible [43
]. Hares affected by myxomatosis were not submitted during the last year, despite the enormous prevalence of this disease in southern Spain, which indicates that this disease has not already seriously affected the hare populations of northern Spain [45
]. The intense lymphoid depletion and lymphocytolysis observed in both diseases may be responsible for the intercurrent enteric and pneumonic disorders detected in these animals [46
The most frequently found cases of toxicoses were associated with the overdose of anticoccidiostatic drugs, mainly monensin and salinomycin, carried out in animals from traditional farms where abusive treatments with these drugs have caused parasitic resistance resulting in higher dosages and causing toxicity problems [47
]. The cases of severe generalized edemas found in some animals in this study were always related to significant liver and kidney degenerative lesions, probably as a result of the damage caused by the use of therapeutic substances.
Neoplasms affected exclusively adult pet rabbits. The increased life expectancy of these animals allows the development of these disorders that are not evidenced in farm animals, or may go unnoticed. The detection frequency and type of tumors were similar to other studies [10
Other less frequently detected conditions were deaths caused by heat stroke, traumas and congenital glaucoma. Lagomorphs, mainly young or pregnant animals, are very sensitive to thermal stress. Above 35 °C, these animals can have difficulty discharging excess heat [50
]. The non-specificity of lesions found makes it necessary to the differential diagnosis with other processes such as RHD, peracute myxomatosis, other peracute septicemia or toxicoses. Rabbits and hares have a long, fragile skeletal systems compared to their heavy musculature and are prone to suffer bone fractures. In this case, most skeletal traumas were associated with road accidents. Finally, glaucoma in rabbits is typically a congenital inherited condition. In our study, cases were detected both in farm and pet rabbits. In farms, animals carrying the defective gene are usually slaughtered since they have small litter sizes and decreased neonatal survival rates [51